Novel securinine derivatives as topoisomerase I based antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2016.06.021

DNA topoisomerase I (Topo I) has been validated as a target for anticancer agents. In this study, a series of novel securinine derivatives bearing β′-hydroxy-α,β-unsaturated ketone moiety were designed and synthesized via a Baylis-Hillman reaction for screening as Topo I inhibitors and antitumor agents. Their topoisomerase I inhibitory activity as well as their cytotoxicity against four human cancer cell lines (A549, HeLa, HepG2, SH-SY5Y) were evaluated, and two pairs of diastereomers 4a-1 and 4a-6 with significant Topo I inhibitory activity and potent anti-proliferative activity against cancer cell lines were identified. The diastereomers were separated, and absolute configurations of five pairs of diastereomers were identified based on X-ray crystallographic analysis and circular dichroism (CD) spectra analysis. Further mechanism studies of the most active compounds 4a-1-R and 4a-1-S indicated that this kind of securinine derivative exhibits a different inhibitory mechanism from that of camptothecin, an established Topo I inhibitor. Unlike camptothecin, compounds 4a-1-R and 4a-1-S specifically inhibits the combination of Topo I and DNA rather than forming the drug-enzyme-DNA covalent ternary complex. In addition, molecular docking and molecular dynamic studies revealed the binding patterns of these compounds with Topo I.

Full text of DOI:10.1016/j.ejmech.2016.06.021

European Journal of Medicinal Chemistry 122 (2016) 149e163
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Novel securinine derivatives as topoisomerase I based antitumor
agents
Wen Hou, Zhen-Ya Wang, Cheng-Kang Peng, Jing Lin*, Xin Liu, Yi-Qun Chang, Jun Xu,
Ren-Wang Jiang, Hui Lin, Ping-Hua Sun, Wei-Min Chen**
College of Pharmacy, Jinan University, Guangzhou 510632, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
DNA topoisomerase I (Topo I) has been validated as a target for anticancer agents. In this study, a series of
novel securinine derivatives bearing
b a,b-unsaturated ketone moiety were designed and
0-hydroxy-
Received 13 November 2015
Received in revised form
6 June 2016
Accepted 13 June 2016
Available online 15 June 2016
synthesized via a Baylis-Hillman reaction for screening as Topo I inhibitors and antitumor agents. Their
topoisomerase I inhibitory activity as well as their cytotoxicity against four human cancer cell lines
(A549, HeLa, HepG2, SH-SY5Y) were evaluated, and two pairs of diastereomers 4a-1 and 4a-6 with
significant Topo I inhibitory activity and potent anti-proliferative activity against cancer cell lines were
identified. The diastereomers were separated, and absolute configurations of five pairs of diastereomers
were identified based on X-ray crystallographic analysis and circular dichroism (CD) spectra analysis.
Further mechanism studies of the most active compounds 4a-1-R and 4a-1-S indicated that this kind of
securinine derivative exhibits a different inhibitory mechanism from that of camptothecin, an estab-
lished Topo I inhibitor. Unlike camptothecin, compounds 4a-1-R and 4a-1-S specifically inhibits the
combination of Topo I and DNA rather than forming the drug-enzyme-DNA covalent ternary complex. In
addition, molecular docking and molecular dynamic studies revealed the binding patterns of these
compounds with Topo I.
Keywords:
Securinine derivatives
Baylis-Hillman reaction
Topoisomerase I
Antitumor agent
© 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction
vinblastine, and camptothecin [5]. Novel Topo I based antitumor
agents with new structural scaffolds are urgently needed to over-
Cancer with annually increasing morbidity has become a major
cause of death in humans. DNA topoisomerase I (Topo I) is an
essential enzyme that catalyze the cleavage and reconnection of
DNA in many key cellular processes such as replication, transcrip-
tion, repair, chromatin assembly, and recombination. Owing to its
high over-expression in cancer cells, Topo I has been recognized as
one of the most promising targets for antitumor drug discovery [1].
Camptothecin (CPT) and its derivatives, topotecan, irinotecan, and
belotecan, well-known Topo I inhibitors have been approved for
clinical treatment of cancer [2e4]. However, some drawbacks of
CPT and its derivatives, such as instability and poor aqueous solu-
bility limit their clinical application. More significantly, increasing
drug resistance and serious side effects severely limit the applica-
tion of natural antitumor compounds such as paclitaxel,
come the defects of CPT and its derivatives. In the last decade, many
other kinds of alkaloids, including indolocarbazoles [6,7], inden-
oisoquinolines [8], dibenzonaphthyridinones [9e11], and aroma-
thecins [12] have been identified as topoisomerase I inhibitors.
Investigating novel alkaloids with new structural scaffolds and
novel mechanisms of action is a potential approach to the discovery
of favorable Topo I based antitumor agents.
It has been reported that securinine, an alkaloid extracted from
securinega [13], shows potent antiproliferative activity against
SW480 (human colon cancer cell line) (IC50 ¼ 25.855
mM) [14,15].
This suggests that securinine may be useful as a lead compound for
the development of antitumor agents. Recent studies have revealed
that
pharmacophore in some anticancer agents [16,17]. Securinine has
an -unsaturated ketone moiety which could be easily converted
to a similar -unsaturated ketone structure by means
0-hydroxy-
of the Baylis-Hillman reaction. In this study, a series of securinine
derivatives containing a -unsaturated ketone moiety
0-hydroxy-
were designed and synthesized in a search for compounds with
b a,b-unsaturated ketone represents a potential
0-hydroxy-
a,b
b
a,b
* Corresponding author.
** Corresponding author.
E-mail addresses: linjing_jnu@163.com (J. Lin), twmchen@jnu.edu.cn
(W.-M. Chen).
b
a,b
http://dx.doi.org/10.1016/j.ejmech.2016.06.021
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

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W. Hou et al. / European Journal of Medicinal Chemistry 122 (2016) 149e163
improved Topo I based antitumor activity. First, the topoisomerase I
inhibitory activity and the cytotoxicity against four human cancer
cell lines (A549, HeLa, HepG2, SH-SY5Y) of the novel securinine
derivatives were evaluated. Then three different electrophoresis
experiments and docking studies were performed in an attempt to
shed light on the inhibitory mechanism of these securinine de-
rivatives on Topo I. In addition, the structure-activity relationships
are discussed. Among the new compounds, several that show sig-
nificant Topo I inhibitory activity and potent anti-proliferative ac-
tivity against cancer cell lines were identified. Mechanism studies
revealed that this kind of securinine derivative enjoys an inhibitory
mechanism different from that of camptothecin.
diastereomers 2a-1, 2a-2, 3a-1, 3a-2, 4a-1, 4a-2, 4a-3, 4a-4, 4a-5,
4a-6, 5a-1 and 5a-2 by HPLC, and the pure isomers 2a-1-S, 2a-1-R,
2a-2-S, 2a-2-R, 3a-1-S, 3a-1-R, 4a-1-S, 4a-1-R, 5a-1-S, 5a-1-R, 3a-
2-a, 3a-2-b, 4a-2-a, 4a-2-b, 4a-3-a, 4a-3-b, 4a-4-a, 4a-4-b, 4a-5-a,
4a-5-b, 4a-6-a, 4a-6-b, 5a-2-a and 5a-2-b were obtained. Pure
compounds whose C-16 configurations were not identified are
named with “-a” (eluted firstly in HPLC) or “-b” (eluted secondly in
HPLC).
The configurations at C-16 in the diastereomers were identified
from X-ray crystallographic analysis of 2a-2-S single crystal. Based
on the absolute structure of 2a-2-S and CD spectra of 2a-2-S and
2a-2-R, the absolute configurations at C-16 in other pure di-
astereomers 2a-1-R, 2a-1-S, 3a-1-R, 3a-1-S, 4a-1-R, 4a-1-S, 5a-1-R
and 5a-1-S were confirmed by recording CD spectra of the di-
astereomers and comparing them with that of 2a-2-S and 2a-2-R.
The crystal structure of compound 2a-2-S with a small Flack
parameter ꢀ0.02(6) indicating the absolute configuration deter-
mined by X-ray crystallography is shown in Fig. 2. Crystal data of
compound 2a-2-S was deposited with the Cambridge Crystallo-
graphic Data Centre (CCDC 1480589).
2. Chemistry
New securinine derivatives shown in Fig. 1 were obtained via
the non-stereoselective, one step Baylis-Hillman reaction [18e20]
between securinine and aromatic aldehydes, which are more
reactive and more susceptible to further derivatization than the
corresponding alkyl aldehydes. Twenty-three securinine de-
rivatives bearing a b a,b-unsaturated ketone moiety and
0-hydroxy-
with different substituents in the phenyl ring, including electron-
withdrawing (Series a) and electron-donating substituents (Series
b) were synthesized. The synthetic routes of compounds 2a-1~2a-
5, 3a-1~3a-5, 4a-1~4a-6, 4b-2~4b-5, 5a-1 and 5a-2 are shown in
Scheme 1. As p-hydroxybenzaldehyde will form a complex with
TiCl4, the three-step route depicted in Scheme 2 was carried out to
obtain compound 4b-1. p-Hydroxy benzaldehyde was reacted with
acetyl chloride [21] to obtain 4-(acetyloxy)- benzaldehyde. Then 4b
was formed via the Baylis-Hillman reaction between 4-(acetyloxy)
benzaldehyde and securinine in the presence of TiCl4 and, finally
4b-1 was obtained by deacetylation of 4b. In order to investigate
the possible impact on the activity of the C-16 configuration of the
chiral carbon bearing the hydroxyl group, we separated
3. Results and discussion
3.1. Topo I inhibitory activity
All synthesized compounds were first screened for their Topo I
inhibitory activity, by assessing the conversion of supercoiled
pBR322 DNA to its relaxed form [22,23] in the presence of the
synthetic securinine derivatives. Camptothecin (CPT), used in the
clinic as a selective Topo I inhibitor, was used as positive control. As
shown in Fig. 3, the supercoiled form (lower bands) increased after
treatment with 100 mM of the securinine derivatives, indicating the
Topo I inhibitory activity of these compounds. A gray scale value
analysis, shown in Table 1, revealed that the inhibition rate was in
Fig. 1. Design of novel securinine derivatives.

W. Hou et al. / European Journal of Medicinal Chemistry 122 (2016) 149e163
151
Scheme 1. Synthesis of securinine derivatives 2a-1~2a-5, 3a-1~3a-5, 4a-1~4a-6, 4b-2~4b-5, 5a-1 and 5a-2.
Scheme 2. Synthesis of compound 4b-1. Reagents and conditions: (i) acetyl chloride, TEA, DCM, 0 ꢁC, 2 h; (ii) securinine, TiCl4, DCM, 2 ꢁC, 12 h; (iii) NaHCO3, 25 ꢁC, 30 min.
Fig. 2. Crystal structure of 2a-2-S.
the range of 24%e56.7%. Notably, all the securinine derivatives are
more effective inhibitors of Topo I than securinine itself, which
structure-activity relationships of the securinine derivatives can be
summarized as follows: (1) the benzyl alcohol group is essential to
the Topo I inhibitory activity; (2) substitution at para-position of
the phenyl ring is better than substitution at ortho- or meta-
position, and m-position substituted compounds show the least
activity; (3) compared to single substitution at para-position,
double substitution both at ortho- and para-positions leads to a
slight decline in activity (5a-1 vs. 4a-1 and 5a-2 vs. 4a-2), which
may be ascribed to the steric hindrance of the active pocket of Topo
I; (4) electron-withdrawing groups at para-position of phenyl ring
are more favorable than electron-donating groups.
shows an inhibition rate of only 23.9% at 100
m
M (Table 1). These
results illustrate that introduction of the a,b
b
0-hydroxy-
-unsatu-
rated ketone moiety into securinine can improve its Topo I inhibi-
tory activity. Among these derivatives, the compounds 4a-1 and 4a-
6 exhibited the highest inhibitory activity, with an inhibition rate of
56.7% and 53.6% respectively (Table 1). Additionally, concentration
gradient experiments further demonstrated the inhibitory effects
of the most effective compound 4a-1 on Topo I inhibition, and the
inhibitory effects were found to be concentration dependent
(Fig. 4). Based on the Topo I inhibitory data, some preliminary
As shown in Scheme 1, the new securinine derivatives contain a

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W. Hou et al. / European Journal of Medicinal Chemistry 122 (2016) 149e163
Fig. 3. Topo I inhibitory activity of securinine and securinine derivatives. Camptothecin (CPT) is a positive control. Supercoiled pBR322 DNA was incubated with Topo I in the
presence or absence of securinine or securinine derivatives at 37 ꢁC for 30 min. Lane A is pBR322 DNA only. Lane B is the mixture of pBR322 DNA and Topo I. Lane C is the mixture of
pBR322 DNA, Topo I and 100 mM CPT. Other lanes are the mixture of pBR322 DNA, Topo I and 100 mM securinine or securinine derivatives.
Table 1
The gray scale value analysis of Fig. 2.
Compounds
Topo I inhibition rate(%)a vs control
Compounds
Topo I inhibition rate(%)a vs control
Camptothecin
Securinine
3a-1
3a-2
3a-3
3a-4
3a-5
4b-1
4b-2
4b-3
4b-4
4b-5
5a-1
68.3b
23.9
36.2
34.9
35.3
34.7
34.7
31.8
31.6
30.9
27.1
24.0
43.1
5a-2
4a-1
4a-2
4a-3
4a-4
4a-5
4a-6
2a-1
2a-2
2a-3
2a-4
2a-5
37.4
56.7
49.8
48.9
47.1
45.7
53.6
40.6
42.3
42.4
39.2
38.4
evaluated by testing the cytotoxicity against four human cancer
cells lines including A549 (adenocarcinoma human alveolar basal
epithelial cell line), HeLa (human cervix tumor cell line), HepG2
(human liver tumor cell), SH-SY5Y (human neuroblastoma cell line)
with an MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-
tetrazolium bromide) assay [24]. Camptothecin was used as posi-
tive control. The cell growth inhibition activities (IC50) of the tested
compounds are shown in Table 2.
As shown in Table 2, most securinine derivatives display
improved cytotoxicity against HeLa, HepG2 and SH-SY5Y cell lines
compared with camptothecin. Notably, compounds 4a-1 and 4a-6
also exhibit the highest cytotoxicity against the four cell lines,
which is consistent with their Topo I inhibitory activity. Moreover, it
was found that all the securinine derivatives are most sensitive to
Fig. 4. Topo I inhibitory activity of different concentration of 4a-1. Supercoiled pBR322
DNA was incubated with Topo I in the presence or absence of 4a-1 at 37 ꢁC for 30 min.
Lane A is pBR322 DNA only. Lane B is the mixture of pBR322 DNA and Topo I. Other
lanes are the mixture of pBR322 DNA, Topo I and 1, 3.125, 6.25, 12.5, 25, 50, 75, 100, 125
mM 4a-1.
chiral carbon generated by Baylis-Hillman reaction, and we firstly
used a mixture of diastereomers in the preliminary screening. To
investigate the possible impact of the configuration of the C-16
chiral carbon bearing the hydroxyl group on the activity, we have
separated the diastereomers 2a-1, 2a-2, 3a-1, 3a-2, 4a-1, 4a-2, 4a-3,
4a-4, 4a-5, 4a-6, 5a-1 and 5a-2 by HPLC and then determined the
Topo I inhibitory activities of the diastereo-pure compounds. As
shown in Fig. 5, the diastereomers did not show any obvious dif-
ference in their activity.
A549 with IC50 values ranging from 1.98 to 11.45 mM, which is 2e11
times greater than that of securinine. In the meantime, the most
active compounds 4a-1 and 4a-6 were further chosen to test their
cytotoxicity against L02 cell line (a healthy mammalian hepato-
cyte), which with an IC50 of 16.71 0.16 mM and 18.23 0.12 mM
respectively. These results turned out that the compounds exhibit
lower toxicity against human L02 hepatocytes compared with that
against cancer cell lines. Especially for A549 cell line, they exhibit
well relative selectivity with a selectivity index ranging from 6.26
to 7.56. Our results indicate that introduction of the b a,b-
0-hydroxy-
3.2. Cytotoxicity
unsaturated ketone moiety into securinine is significant, improving
the Topo I inhibitory activity as well as the antitumor activity.
The antitumor activity of all synthesized compounds was then

W. Hou et al. / European Journal of Medicinal Chemistry 122 (2016) 149e163
153
Fig. 5. (A) Topo I inhibitory activities of the pure isomers of the most active 4a series and the representative compounds of the other three series. Camptothecin (CPT) is a positive
control. Supercoiled pBR322 DNA was incubated with Topo I in the presence or absence of compound at 37 ꢁC for 30 min. Lane A is pBR322 DNA only. Lane B is the mixture of
pBR322 DNA and Topo I. Lane C is the mixture of pBR322 DNA, Topo I and 100
value analysis of A.
mM CPT. Other lanes are the mixture of pBR322 DNA, Topo I and 100 mM compound; (B) The gray scale
Additionally, consistent with the Topo I inhibition results, single
electrophilic substitution at para-position is most favorable for the
activity, and meta-position substitution (as in 4b-1e4b-5) is unfa-
vorable. The similar cell cytotoxicities of the pure isomers 4a-1-S
and 4a-1-R, 4a-2-a and 4a-2-b, 4a-3-a and 4a-3-b, 4a-4-a and 4a-
4-b, 4a-5-a and 4a-5-b, 4a-6-a and 4a-6-b confirm that the
configuration of the chiral carbon has no significant effect on their
bioactivity.
insertion.
3.4. Topo I-mediated DNA cleavage assay
To determine if our compounds can act as Topo I inhibitors by
stabilizing the Topo I-DNA cleavage complex through formation of
drug-enzyme-DNA covalent ternary complex, similar to CPT [27], a
Topo I-mediated DNA cleavage assay was performed. Pure com-
pounds 4a-1-S and 4a-1-R were used to test this possible mecha-
nism and CPT was used as positive control. In this experiment,
formation of a drug-enzyme-DNA covalent ternary complex of CPT
will result in an increase in linear DNA (the upper bands in Fig. 8),
which moves most slowly in the agarose gel. As displayed in Fig. 8,
when treated with 25
as expected. Treatment with compound 4a-1-S and 4a-1-R failed to
induce the generation of linear DNA at even 200 M. These results
reveal that, unlike camptothecin, our compounds exhibit a different
mechanism rather than stabilizing the Topo I-DNA cleavage com-
plex by forming the drug-enzyme-DNA covalent ternary complex.
3.3. DNA-insertion assay
The Topo I inhibition assay well established that Topo I is a
target of securinine derivatives. The catalytic process of Topo I [25]
is vividly depicted in Fig. 6 which indicates that four possible
mechanism is valid for Topo I inhibitors, namely through inserting
into DNA, forming drug-enzyme-DNA covalent or noncovalent
ternary complex, and retarding the combination between enzyme
and DNA. To demonstrate the possible mechanism of Topo I
inhibitory effects of this type of compounds, a DNA-insertion assay
[26] was firstly conducted to investigate the DNA intercalating
ability of these compounds. Ethidium bromide (EB), a DNA inter-
calating agent, was used as a positive control, and CPT, which in-
hibits Topo I by forming the drug-enzyme-DNA covalent ternary
complex, was used as a negative control. This experiment uses a
large excess of Topo I, so that only a DNA intercalating agent such as
EB can inhibit the Topo I-mediated conversion of the supercoiled
form (lower bands) to the relaxed form (upper bands). As shown in
Fig. 7, after pre-incubation with EB, the relaxation ability of Topo I
was obscured, but it still could convert the supercoiled DNA to the
relaxed form after the pre-incubation with our compounds and
CPT. This result demonstrates that, similar to CPT, these securinine
derivatives fail to exhibit Topo I inhibitory activity through DNA
mM and 50 mM CPT, linear DNA was generated
m
3.5. Inhibition of the combination between DNA and Topo I
The Topo I-mediated DNA cleavage assay suggested that 4a-1-S
and 4a-1-R might inhibit Topo I prior to Topo I-mediated DNA
cleavage. We therefore investigated whether 4a-1-S and 4a-1-R
could interfere with the binding of Topo I to DNA using an elec-
trophoretic mobility shift assay (EMSA) [28,29]. In this experiment
CPT, which inhibits the religation of Topo I without interfering with
the binding of Topo I to DNA, was used as negative control. As
shown in Fig. 9, CPT as expected, has no effect on the formation of
the Topo I-DNA complex (the middle bands), but when compounds
4a-1-S and 4a-1-R were added, the TopoI-DNA complex diminished

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W. Hou et al. / European Journal of Medicinal Chemistry 122 (2016) 149e163
Table 2
Cytotoxicity of securinine and its derivatives against cancer cell lines.
Compounds IC50
M)a
A549
Camptothecinb 0.12 0.04
(
m
HeLa
HepG2
SH-SY5Y
59.13 0.02 29.81 0.09
>100
Securinine
2a-1
2a-2
2a-3
2a-4
2a-5
3a-1
3a-2
3a-3
23.59 0.03 50.23 0.11 36.87 0.04
78.90 0.13
8.69 0.04
22.01 0.13
19.90 0.22
23.11 0.17
21.93 0.20
8.70 0.21
27.10 0.09
27.81 0.12
20.18 0.21
24.85 0.32
8.50 0.14
8.52 0.03
8.48 0.08
19.80 0.28
19.12 0.15
19.00 0.16
20.00 0.12
20.10 0.10
19.90 0.25
22.30 0.09
22.40 0.16
22.28 0.17
18.90 0.13
18.91 0.12
18.87 0.17
8.57 0.10
8.27 0.11
8.62 0.07
23.01 0.07
22.47 0.26
21.04 0.09
15.12 0.12
3.91 0.17
6.89 0.08
7.01 0.03
7.11 0.18
7.01 0.16
4.04 0.22
8.01 0.12
8.11 0.09
6.78 0.16
13.01 0.18 10.09 0.11
10.40 0.12 9.16 0.17
12.70 0.16 12.61 0.08
14.9 0.18
9.98 0.13
11.08 0.10
6.77 0.09
16.77 0.09 16.11 0.18
11.08 0.20 15.60 0.15 17.80 0.13
19.09 0.21 15.11 0.08
3a-4
3a-5
4a-1
9.01 0.10
7.08 0.11
2.21 0.06
1.98 0.08
2.23 0.11
5.01 0.02
4.95 0.10
5.09 0.15
6.21 0.09
6.18 0.13
6.20 0.11
6.32 0.10
6.24 0.12
6.30 0.20
6.22 0.15
6.20 0.16
6.22 0.17
2.91 0.08
2.78 0.21
3.01 0.16
6.99 0.05
15.6 0.07
8.26 0.01
7.99 0.02
8.00 0.20
17.90 0.06
4.13 0.03
4.01 0.05
4.19 0.09
4a-1-S
4a-1-R
4a-2
4a-2-a
4a-2-b
4a-3
4a-3-a
4a-3-b
4a-4
4a-4-a
4a-4-b
4a-5
4a-5-a
4a-5-b
4a-6
4a-6-a
4a-6-b
4b-1
12.77 0.09 10.08 0.06
12.80 0.12 10.00 0.10
12.66 0.09 10.06 0.12
12.98 0.08 13.04 0.05
12.50 0.11 13.12 0.08
12.48 0.17 13.12 0.14
12.78 0.13 14.10 0.17
12.79 0.11 14.10 0.09
12.78 0.08 14.11 0.13
13.00 0.12 14.19 0.02
13.09 0.11 14.10 0.14
12.08 0.12 14.11 0.19
Fig. 6. The relaxation process of Topo I for supercoiled DNA and mechanism of Topo I
inhibitors including CPT and 4a-1.
8.99 0.11
8.80 0.09
9.08 0.13
2.42 0.08
2.23 0.12
2.57 0.19
18.89 0.12 17.06 0.13
10.04 0.13 10.02 0.03 17.80 0.02
4b-2
4b-3
4b-4
4b-5
9.05 0.17
8.81 0.05
7.05 0.11
17.80 0.11 16.7 0.05
9.07 0.02 7.09 0.11
21.44 0.06 20.09 0.001 25.41 0.09
Fig. 7. DNA-insertion assay of pure compounds of 4a series. Ethidium bromide (EB) is a
positive control. Supercoiled pBR322 DNA was incubated with Topo I in the presence or
absence of EB, CPT, and the tested compounds at 37 ꢁC for 30 min. Lane A is pBR322
DNA only. Lane B is the mixture of pBR322 DNA and Topo I. Lane C is the mixture of
5a-1
5a-2
11.45 0.10 7.11 0.04
5.18 0.12 14.90 0.21 13.56 0.23
5.98 0.01
8.71 0.13
22.10 0.09
a
All dates represent mean
S.D. from different experiments performed in
triplicate.
pBR322 DNA, Topo I and 100
25 M EB. Other lanes are the mixture of pBR322 DNA, Topo I and 100
compounds.
m
M CPT. Lane E is the mixture of pBR322 DNA, Topo I and
b
Camptothecin acted as a positive control for MTT assay.
m
mM tested
linearly in a concentration-dependent manner. These EMSA results
clearly indicate that the Topo I inhibitory mechanism of our com-
pounds is that specifically inhibits the combination of Topo I and
DNA. This is an inhibitory mechanism completely different from
that of CPT. Moreover, given the EMSA and DNA-insertion assay
results we can speculate that the synthetic compounds are likely to
interact with Topo I, but not with DNA, thus interfering with the
binding of Topo I to DNA.
pocket of Topo I by forming hydrogen bonds (the hydrogen bonds
were showed as red lines) with different amino acid residue in the
pocket of Topo I (4a-1-R with ARG364, ASP533, and ASN722. 4a-1-S
with LYS532, THR718, and ASN722), indicating the satisfactory
integration of 4a-1-S and 4a-1-R with Topo I. Our results supported
the strong binding of 4a-1-S and 4a-1-R to Topo I, providing evi-
dence for the hypothesis that the Topo I-DNA combination inter-
ference effects result from the binding of the compound with Topo
I.
3.6. Molecular modeling
Since the mechanism studies revealed that this class of com-
pounds apparently blocks the combination between DNA and Topo
I by interaction with Topo I, a molecular docking study was per-
formed to confirm this hypothesis and to better understand the
possible interaction mode of these compounds with Topo I. The
structure of Topo I used in the docking studies was obtained from
the Protein Data Bank (PDB ID: 1K4T) [30], then 4a-1-S and 4b-1-R
were chosen for the docking analysis. The original complexes were
generated by SYBYL-8.1 Surflex-dock and then validated by mo-
lecular dynamics (MD) simulation (Fig. S1). The average minimized
4. Conclusion
In this study, a series of novel securinine derivatives bearing a
b
0-hydroxy-
a,b-unsaturated ketone moiety, which is a pharmaco-
phore with potential antitumor activity, were designed, synthe-
sized and evaluated for Topo I inhibitory activity and cytotoxicity
against four human cancer cell lines. Among the synthesized
securinine derivatives, some exhibited promising Topo I inhibitory
activity as well as cytotoxicity against cancer cell lines, especially
for the A549 cell line. All the securinine derivatives were found to
be more effective than securinine itself both in the inhibition of
Topo I activity and inhibition the growth of cancer cell lines. This
structures, whose maximum free energy (DG) calculated by MM-
PBSA were ꢀ34.87 kcal/mol (4a-1-R) and ꢀ33.06 kcal/mol (4a-1-
S), were used for binding mode analysis. As indicated in Fig.10, both
R (gray) and S (blue) configurations of 4a-1 fit well into the active
showed that introduction of the
tone moiety into securinine is
b
0-hydroxy-
feasible strategy for the
a,b-unsaturated ke-
a

W. Hou et al. / European Journal of Medicinal Chemistry 122 (2016) 149e163
155
Fig. 8. (A) Topo I-mediated DNA cleavage assay of pure compounds 4a-1-S and 4a-1-R. Camptothecin (CPT) is a positive control. Lane A is pBR322 DNA only. Lane B is the mixture of
pBR322 DNA and Topo I. Other lanes are the mixture of pBR322 DNA, Topo I and 25, 50 M CPT or 25, 50, 100, 200 M 4a-1-S and 4a-1-R; (B) The gray scale value analysis of A.
m
m
Fig. 9. (A) EMSA-assay of pure compounds 4a-1-S and 4a-1-R. Camptothecin (CPT) is a negative control. Lane A is pBR322 DNA only. Lane B is the mixture of pBR322 DNA and Topo
I. Line C is the mixture of pBR322 DNA, Topo I and 50 or 100
scale value analysis of A.
mM CPT. Other lanes are the mixture of pBR322 DNA, Topo I and 50, 100, 200, or 300 mM 4a-1-S and 4a-1-R; (B) The gray
development of securinine derivatives as Topo I-based antitumor
agents. Further mechanistic and docking studies showed that this
kind of securinine related compounds exhibit a different inhibitory
mechanism from that of CPT. Unlike CPT, the Topo I inhibitory ac-
tivity of the synthesized compounds was due at least in part, to
their interaction with Topo I, and interference in the interaction of
Topo I with DNA as depicted in Fig. 6. Since our securinine de-
rivatives do not covalently bind to Topo I, they represent a class of
Topo I inhibitors with relatively low toxicity as compared to CPT.

156
W. Hou et al. / European Journal of Medicinal Chemistry 122 (2016) 149e163
Fig. 10. (A) Binding model between 4a-1-R and Topo I from docking and molecular dynamics; (B) Binding model between 4a-1-S and Topo I from docking and molecular dynamics.
5. Experiment section
369.1445.
5.1. Chemistry part
5.1.1.2. (6S,11aR,11Bs,16R)-3-(Hydroxy(2-nitrophenyl)methyl)-
6,8,9,10,11,11a-hexahydro-2H-6,11b-methanofuro [2,3-c]pyrido [1,2-
a]azepin-2-one (2a-1-R). Yellow solid. m.p. 178.1e179.3 ꢁC; 1H
All chemicals were purchased from Alfa Aesar or Sigma Aldrich
Co. Intermediate 4b was synthesized according to published
methods [21]. The synthetic routes to the twenty-three securinine
derivatives are shown in Scheme 1 and Scheme 2. All the com-
pounds were fully identified through 1H and 13C-nuclear magnetic
resonance (NMR), mass spectrometry (MS) and high resolution
mass spectrometry (ESI-HRMS) before biological testing.
NMR (300 MHz, CDCl3)
d
8.30 (t, J ¼ 1.7 Hz, 1H), 8.17 (dd, J ¼ 8.2,
1.3 Hz, 1H), 7.81 (d, J ¼ 7.7 Hz, 1H), 7.55 (t, J ¼ 7.9 Hz, 1H), 6.56 (d,
J ¼ 9.2 Hz, 1H), 6.40 (dd, J ¼ 9.3, 5.3 Hz, 1H), 5.77 (s, 1H, 16-H), 3.82
(t, J ¼ 4.6 Hz, 1H), 2.96 (dt, J ¼ 10.6, 3.8 Hz, 1H), 2.50 (dd, J ¼ 9.3,
4.1 Hz, 1H), 2.43 (dd, J ¼ 9.5, 6.2 Hz, 1H), 2.16 (d, J ¼ 9.2 Hz, 1H),
1.92e1.85 (m, 1H), 1.75 (d, J ¼ 9.3 Hz, 1H), 1.69e1.52 (m, 4H), 1.26 (s,
1H).
5.1.1. General procedure of the preparation of series a and series b
except 4b-1
5.1.1.3. (6S,11aR,11Bs,16S)-3-(Hydroxy(2-nitrophenyl)methyl)-
6,8,9,10,11,11a-hexahydro-2H-6,11b-methanofuro [2,3-c]pyrido [1,2-
a]azepin-2-one (2a-1-S). Yellow solid. m.p. 165.4e166.3 ꢁC; 1H
Securinine (0.69 mmol, 150 mg) and
a benzaldehyde
(1.04 mmol) were added into a dry anaerobic double-neck flask
containing 5 ml dry DCM at 2 ꢁC. When these were completely
dissolved, TiCl4 (1.248 mmol) was added. The reaction was moni-
tored by TLC on an hourly basis and was terminated after 12 h.
Saturated sodium bicarbonate solution was added until a pH of 9.0
was observed. The mixture was filtered, and extracted with DCM.
The DCM layer was dried over anhydrous Na2SO4, filtered and
concentrated to obtain a residue, which was purified through silica
gel column (DCM: MeOH ¼ 200:1) to give the compounds in series
a and series b, with the exception of 4b-1. Diastereomers 2a-1, 2a-2,
3a-1, 3a-2, 4a-1, 4a-2, 4a-3, 4a-4, 4a-5, 4a-6, 5a-1 and 5a-2 were
separated by HPLC equipped with UltiMate3000 and an Ultimate
Cosmosil Pecked 5C-C18 column (20 I. D. ꢂ 250 mm) which was
eluted with methanol - water (75:25e45:55, for 2a-1, 2a-2, 3a-1,
3a-2, 4a-1, 4a-2, 4a-3, 4a-4, 4a-5, 4a-6, and 5a-2) and an 00G-
NMR (300 MHz, CDCl3)
d 8.28 (s, 1H), 8.20e8.13 (m, 1H), 7.80 (d,
J ¼ 7.7 Hz, 1H), 7.56 (t, J ¼ 7.9 Hz, 1H), 6.46 (d, J ¼ 8.7 Hz, 1H), 6.39
(dd, J ¼ 9.3, 5.0 Hz, 1H), 5.78 (s, 1H, 16-H), 3.82 (t, J ¼ 4.5 Hz, 1H),
2.95 (dt, J ¼ 10.5, 3.7 Hz, 1H), 2.51 (dd, J ¼ 9.3, 4.1 Hz,1H), 2.45e2.34
(m, 1H), 2.06 (dd, J ¼ 10.9, 2.8 Hz, 1H), 1.91e1.82 (m, 1H), 1.78 (d,
J ¼ 9.3 Hz, 1H), 1.65e1.48 (m, 4H), 1.25 (s, 1H).
5.1.1.4. (6S,11aR,11bS)-3-((2-Fluorophenyl)(hydroxy)methyl)-
6,8,9,10,11,11a-hexahydro-2H-6,11b-methanofuro [2,3-c]pyrido [1,2-
a]azepin-2-one (2a-2). Yellow solid, yield 75% (R:S ¼ 1.58:1); 1H
NMR (300 MHz, CDCl3) d 7.66e7.54 (m, 1H), 7.35e7.25 (m, 1H), 7.19
(t, J ¼ 7.5 Hz, 1H), 7.07e6.98 (m, 1H), 6.39e6.23 (m, 2H), 5.93 (d,
J ¼ 4.9 Hz,1H), 3.82e3.72 (m,1H), 2.98e2.87 (m,1H), 2.54e2.32 (m,
2H), 2.15e2.00 (m, 1H), 1.92e1.81 (m, 1H), 1.80e1.69 (m, 1H),
1.67e1.43 (m, 4H), 1.35e1.15 (m, 1H). 13C NMR (75 MHz, CDCl3)
4459-E0 Lux 5
m
m Cellulose-1, LC column (250 ꢂ 4.6 mm) which
was eluted with acetonitrile - water (55:45, for 5a-1) containing
0.1% DEA at a flow rate of 4 mL/min, giving pure isomers 2a-1-S, 2a-
1-R, 2a-2-S, 2a-2-R, 3a-1-S, 3a-1-R, 4a-1-S, 4a-1-R, 5a-1-S, 5a-1-R,
3a-2-a, 3a-2-b, 4a-2-a, 4a-2-b 4a-3-a, 4a-3-b, 4a-4-a, 4a-4-b, 4a-5-
a, 4a-5-b, 4a-6-a, 4a-6-b, 5a-2-a and 5a-2-b.
d
173.58, 173.50, 163.43, 163.24, 161.58, 161.44, 158.31, 158.17,
139.93,139.76,129.89,129.85,129.78,129.75,128.10,128.05,124.65,
124.61, 124.57, 120.63, 120.48, 116.80, 116.72, 115.67, 115.39, 88.78,
88.76, 63.34, 63.29, 63.18, 62.97, 58.82, 48.83, 48.78, 42.49, 42.23,
27.38, 27.32, 25.92, 24.52, 24.45; ESI-LRMS m/z: 342.4 [MþH]þ; ESI-
HRMS m/z: 342.1502 [MþH]þ, calcd for C20H21FNO3 342.1500.
5.1.1.1. (6S,11aR,11bS)-3-(Hydroxy(2-nitrophenyl)methyl)-
6,8,9,10,11,11a-hexahydro-2H-6,11b-methanofuro [2,3-c]pyrido [1,2-
a]azepin-2-one (2a-1). Yellow solid, yield 78% (R:S ¼ 1:1.82); 1H
5.1.1.5. (6S,11aR,11bS,16S)-3-((2-Fluorophenyl)(hydroxy)methyl)-
6,8,9,10,11,11a-hexahydro-2H-6,11b-methanofuro [2,3-c]pyrido [1,2-
a]azepin-2-one (2a-2-S). Yellow solid. m.p. 169.0e169.1 ꢁC; 1H
NMR (300 MHz, CDCl3)
d 7.99e7.87 (m, 2H), 7.71e7.61 (m, 1H),
NMR (300 MHz, CDCl3)
d
7.59 (td, J ¼ 7.6, 1.7 Hz, 1H), 7.31 (tdd,
7.52e7.42 (m, 1H), 6.26e6.18 (m, 2H), 6.13e5.99 (m, 1H), 3.73 (t,
J ¼ 4.6 Hz, 1H), 2.88 (dt, J ¼ 10.4, 3.7 Hz, 1H), 2.46e2.27 (m, 2H),
2.08e1.95 (m, 1H), 1.87e1.74 (m, 1H), 1.69 (dd, J ¼ 9.2, 3.7 Hz, 1H),
1.62e1.38 (m, 4H), 1.31e1.07 (m, 1H). 13C NMR (75 MHz, CDCl3)
J ¼ 7.3, 5.3, 1.8 Hz, 1H), 7.20 (td, J ¼ 7.5, 1.1 Hz, 1H), 7.08e7.00 (m,
1H), 6.38e6.27 (m, 2H), 5.94 (s, 1H,16-H), 3.78 (t, J ¼ 4.4 Hz, 1H),
2.93 (dt, J ¼ 10.5, 3.7 Hz,1H), 2.49 (dd, J ¼ 9.2, 4.1 Hz,1H), 2.44e2.32
(m, 1H), 2.05 (dd, J ¼ 11.0, 2.7 Hz, 1H), 1.90e1.75 (m, 2H), 1.63e1.47
(m, 4H), 1.24 (s, 1H).
d
173.26, 173.21, 164.31, 164.08, 147.76, 140.22, 136.24, 136.05,
133.70, 133.59, 129.04, 128.89, 128.85, 124.75, 120.38, 120.28,
116.84, 116.72, 88.60, 88.57, 64.10, 63.16, 62.82, 58.71, 58.66, 48.67,
42.50, 41.87, 27.07, 25.70, 25.66, 24.29; ESI-LRMS m/z: 369.3
[MþH]þ; ESI-HRMS m/z: 369.1441 [MþH]þ, calcd for C20H21N2O5
5.1.1.6. (6S,11aR,11bS,16R)-3-((2-Fluorophenyl)(hydroxy)methyl)-
6,8,9,10,11,11a-hexahydro-2H-6,11b-methanofuro [2,3-c]pyrido [1,2-

W. Hou et al. / European Journal of Medicinal Chemistry 122 (2016) 149e163
157
a]azepin-2-one (2a-2-R). Yellow solid. m.p. 158.2e159.1 ꢁC; 1H
NMR (300 MHz, CDCl3)
J ¼ 7.9, 6.4, 2.6 Hz, 1H), 7.19 (td, J ¼ 7.5, 1.1 Hz, 1H), 7.10e6.98 (m,
1H), 6.36e6.23 (m, 2H), 5.95 (s, 1H,16-H), 3.77 (dd, J ¼ 6.0, 3.7 Hz,
1H), 2.93 (dt, J ¼ 10.5, 3.7 Hz, 1H), 2.45 (dd, J ¼ 9.3, 4.2 Hz, 1H), 2.39
(dd, J ¼ 7.3, 3.7 Hz, 1H), 2.09 (dd, J ¼ 11.2, 2.5 Hz, 1H), 1.92e1.81 (m,
1H), 1.73 (d, J ¼ 9.2 Hz, 1H), 1.68e1.47 (m, 4H), 1.32e1.17 (m, 1H).
C20H21N2O5 369.1445.
d
7.61 (td, J ¼ 7.6, 1.7 Hz, 1H), 7.30 (ddd,
5.1.1.11. (6S,11aR,11bS,16R)-3-(Hydroxy(3-nitrophenyl)methyl)-
6,8,9,10,11,11a-hexahydro-2H-6,11b-methanofuro [2,3-c]pyrido [1,2-
a]azepin-2-one (3a-1-R). Yellow solid. m.p. 174.0e171.1 ꢁC; 1H
NMR (300 MHz, CDCl3)
d
8.32e8.29 (m, 1H), 8.18 (dd, J ¼ 8.6, 1.8 Hz,
1H), 7.84e7.79 (m, 1H), 7.56 (t, J ¼ 8.0 Hz, 1H), 6.54 (d, J ¼ 9.3 Hz,
1H), 6.40 (dd, J ¼ 9.2, 5.3 Hz, 1H), 5.78 (s, 1H,16-H), 3.82 (t,
J ¼ 4.5 Hz, 1H), 2.96 (dd, J ¼ 6.3, 4.0 Hz, 1H), 2.50 (dd, J ¼ 9.3, 4.1 Hz,
1H), 2.47e2.39 (m, 1H), 2.17 (s, 1H), 2.15 (d, J ¼ 3.4 Hz, 1H), 1.88 (dd,
J ¼ 10.4, 4.1 Hz, 1H), 1.66e1.60 (m, 4H), 1.27 (s, 1H).
5.1.1.7. (6S,11aR,11bS)-3-((2-Chlorophenyl)(hydroxy)methyl)-
6,8,9,10,11,11a-hexahydro-2H-6,11b-methanofuro [2,3-c]pyrido [1,2-
a]azepin-2-one (2a-3). Yellow solid, yield 76%; 1H NMR
(300 MHz, CDCl3) d 7.78e7.68 (m, 1H), 7.39e7.26 (m, 3H), 6.27e6.17
(m, 1H), 6.02e5.89 (m, 2H), 3.75 (dd, J ¼ 8.6, 4.1 Hz, 1H), 2.91 (dt,
J ¼ 10.4, 3.8 Hz, 1H), 2.53e2.31 (m, 2H), 2.13e2.02 (m, 1H),
1.92e1.81 (m, 1H), 1.76 (t, J ¼ 9.8 Hz, 1H), 1.68e1.47 (m, 4H),
5.1.1.12. (6S,11aR,11bS,16S)-3-(Hydroxy(3-nitrophenyl)methyl)-
6,8,9,10,11,11a-hexahydro-2H-6,11b-methanofuro [2,3-c]pyrido [1,2-
a]azepin-2-one (3a-1-S). Yellow solid. m.p. 183.4e184.5 ꢁC; 1H
1.29e1.19 (m,1H). 13C NMR (75 MHz, CDCl3)
d
173.84,173.74,163.76,
NMR (300 MHz, CDCl3)
d
8.28 (d, J ¼ 1.5 Hz, 1H), 8.18 (d, J ¼ 8.8 Hz,
163.58, 139.95, 139.75, 138.16, 132.41, 132.20, 129.70, 129.66, 129.38,
128.29, 128.11, 127.36, 127.30, 120.55, 120.43, 116.70, 116.67, 88.78,
88.73, 65.54, 63.17, 62.93, 58.80, 48.77, 42.54, 42.05, 27.32, 27.25,
25.84, 24.47, 24.40; ESI-LRMS m/z: 358.3 [MþH]þ; ESI-HRMS m/z:
358.1213 [MþH]þ, calcd for C20H21ClNO3 358.1204.
1H), 7.81 (d, J ¼ 7.7 Hz, 1H), 7.57 (t, J ¼ 8.0 Hz, 1H), 6.50e6.41 (m,
2H), 5.79 (s, 1H, 16-H), 3.89e3.82 (m, 1H), 3.03e2.91 (m, 1H), 2.54
(dd, J ¼ 9.4, 3.8 Hz, 1H), 2.46 (s, 1H), 2.17 (s, 1H), 1.89 (s, 1H), 1.81 (d,
J ¼ 9.4 Hz, 1H), 1.65e1.58 (m, 4H), 1.25 (s, 1H).
5.1.1.13. (6S,11aR,11bS)-3-((3-Fluorophenyl)(hydroxy)methyl)-
6,8,9,10,11,11a-hexahydro-2H-6,11b-methanofuro [2,3-c]pyrido [1,2-
a]azepin-2-one (3a-2). Yellow solid, yield 68% (a:b ¼ 1:3.49); 1H
5.1.1.8. (6S,11aR,11bS)-3-((2-Bromophenyl)(hydroxy)methyl)-
6,8,9,10,11,11a-hexahydro-2H-6,11b-methanofuro [2,3-c]pyrido [1,2-
a]azepin-2-one (2a-4). Yellow solid, yield 74%; 1H NMR
NMR (300 MHz, CDCl3)
d
7.39e7.27 (m, 1H), 7.17 (t, J ¼ 9.9 Hz, 2H),
(300 MHz, CDCl3)
d
7.78e7.70 (m, 1H), 7.59e7.51 (m, 1H), 7.40 (t,
7.05e6.94 (m, 1H), 6.48e6.29 (m, 2H), 5.66 (d, J ¼ 3.2 Hz, 1H),
3.86e3.74 (m, 1H), 2.94 (dt, J ¼ 10.5, 3.8 Hz, 1H), 2.57e2.33 (m, 2H),
2.20e2.01 (m, 1H), 1.86 (d, J ¼ 13.3 Hz, 1H), 1.75 (t, J ¼ 10.0 Hz, 1H),
1.68e1.45 (m, 4H), 1.28e1.23 (m, 1H). 13C NMR (75 MHz, CDCl3)
J ¼ 7.6 Hz, 1H), 7.21 (t, J ¼ 7.7 Hz, 1H), 6.26e6.10 (m, 1H), 5.93 (d,
J ¼ 3.9 Hz,1H), 5.81 (dd, J ¼ 12.7, 9.3 Hz,1H), 3.74 (dd, J ¼ 8.4, 4.1 Hz,
1H), 2.91 (dt, J ¼ 10.3, 3.8 Hz, 1H), 2.52e2.28 (m, 2H), 2.06 (d,
J ¼ 11.0 Hz, 1H), 1.85 (dd, J ¼ 8.4, 4.6 Hz, 1H), 1.80e1.72 (m, 1H),
1.68e1.48 (m, 4H), 1.33e1.19 (m, 1H). 13C NMR (75 MHz, CDCl3)
d
173.47, 173.34, 164.75, 163.47, 163.37, 161.48, 161.44, 143.89,
143.79, 140.18, 140.13, 130.44, 130.33, 122.05, 121.93, 120.92, 120.54,
115.25, 115.02, 113.53, 113.48, 113.23, 113.18, 88.77, 88.71, 68.82,
68.41, 63.11, 63.06, 58.88, 58.83, 48.77, 42.31, 42.24, 27.30, 27.18,
25.82, 25.70, 24.37, 24.28; ESI-LRMS m/z: 342.3 [MþH]þ; ESI-HRMS
m/z: 342.1505 [MþH]þ, calcd for C20H21FNO3 342.1507.
d
173.97, 173.83, 163.82, 163.59, 139.94, 139.77, 139.71, 139.69,
133.04, 132.98, 129.73, 128.59, 128.42, 127.98, 127.91, 122.50, 122.24,
120.57,120.43,116.76,116.73, 88.81, 88.75, 67.72, 63.17, 62.95, 58.81,
48.81, 42.05, 27.39, 27.31, 25.92, 25.84, 24.56, 24.48; ESI-LRMS m/z:
402. [MþH]þ; ESI-HRMS m/z: 402.0697 [MþH]þ, calcd for
C
20H21BrNO3 402.0699.
5.1.1.14. (6S,11aR,11bS,)-3-((3-Fluorophenyl)(hydroxy)methyl)-
6,8,9,10,11,11a-hexahydro-2H-6,11b-methanofuro [2,3-c]pyrido [1,2-
a]azepin-2-one (3a-2-a). Yellow solid. m.p. 167.0e168.0 ꢁC; 1H
5.1.1.9. 2-(Hydroxy((6S,11aR,11bS)-2-oxo-6,8,9,10,11,11a-hexahydro-
2H-6,11b-methanofuro [2,3-c]pyrido [1,2-a]azepin-3-yl)methyl)ben-
zonitrile (2a-5). Yellow solid, yield 70%; 1H NMR (300 MHz, CDCl3)
NMR (300 MHz, CDCl3) d 7.38e7.29 (m, 1H), 7.23e7.13 (m, 2H), 7.00
(td, J ¼ 8.8, 2.9 Hz, 1H), 6.41 (d, J ¼ 8.7 Hz, 1H), 6.33 (dd, J ¼ 9.3,
5.2 Hz, 1H), 5.67 (s, 1H, 16-H), 3.79 (t, J ¼ 4.6 Hz, 1H), 2.94 (dt,
J ¼ 10.4, 3.7 Hz, 1H), 2.49 (dd, J ¼ 9.2, 4.0 Hz, 1H), 2.46e2.38 (m, 1H),
2.14 (dd, J ¼ 11.1, 2.3 Hz, 1H), 1.93e1.82 (m, 1H), 1.74 (d, J ¼ 9.3 Hz,
1H), 1.67e1.50 (m, 4H), 1.25 (s, 1H).
d
7.85 (t, J ¼ 7.2 Hz, 1H), 7.67e7.41 (m, 3H), 6.45e5.95 (m, 2H), 5.61
(s, 1H), 3.78e3.60 (m, 1H), 2.86 (s, 1H), 2.50e2.36 (m, 1H), 2.29 (s,
1H), 2.16e1.68 (m, 3H), 1.67e1.41 (m, 4H), 1.25e1.16 (m, 1H). 13C
NMR (75 MHz, CDCl3)
d 173.34, 173.20, 171.19, 170.92, 164.48,
164.46, 145.79, 145.40, 140.79, 140.62, 132.71, 131.31, 130.81, 128.96,
128.91, 124.09, 124.03, 123.52, 120.00, 113.85, 113.77, 88.75, 88.72,
63.09, 62.90, 58.63, 58.39, 52.41, 52.11, 48.75, 48.69, 42.50, 42.34,
27.31, 25.85, 24.44; ESI-LRMS m/z: 349.3 [MþH]þ; ESI-HRMS m/z:
349.1544 [MþH]þ, calcd for C21H21N2O3 349.1547.
5.1.1.15. (6S,11aR,11bS,)-3-((3-Fluorophenyl)(hydroxy)methyl)-
6,8,9,10,11,11a-hexahydro-2H-6,11b-methanofuro [2,3-c]pyrido [1,2-
a]azepin-2-one (3a-2-b). Yellow solid. m.p. 155.6e156.6 ꢁC; 1H
NMR (300 MHz, CDCl3)
d
7.34 (td, J ¼ 7.9, 5.8 Hz, 1H), 7.16 (dd,
J ¼ 14.7, 5.0 Hz, 2H), 7.01 (td, J ¼ 8.6, 2.3 Hz, 1H), 6.38e6.27 (m, 2H),
5.68 (s, 1H, 16-H), 3.79 (t, J ¼ 3.7 Hz, 1H), 2.94 (dt, J ¼ 10.5, 3.7 Hz,
1H), 2.50 (dd, J ¼ 9.3, 4.1 Hz, 1H), 2.45e2.33 (m, 1H), 2.06 (dd,
J ¼ 10.9, 2.4 Hz, 1H), 1.92e1.82 (m, 1H), 1.77 (d, J ¼ 9.3 Hz, 1H),
1.65e1.48 (m, 4H), 1.25 (s, 1H).
5.1.1.10. (6S,11aR,11bS)-3-(Hydroxy(3-nitrophenyl)methyl)-
6,8,9,10,11,11a-hexahydro-2H-6,11b-methanofuro [2,3-c]pyrido [1,2-
a]azepin-2-one (3a-1). Yellow solid, yield 70% (R:S ¼ 1:1.23); 1H
NMR (300 MHz, CDCl3)
d
8.28 (d, J ¼ 1.8 Hz, 1H), 8.18e8.10 (m, 1H),
7.78 (d, J ¼ 7.7 Hz, 1H), 7.53 (td, J ¼ 8.0, 3.9 Hz, 1H), 6.65e6.45 (m,
1H), 6.38 (dd, J ¼ 9.3, 5.2 Hz, 1H), 5.77 (s, 1H), 3.80 (dd, J ¼ 9.8,
5.0 Hz, 1H), 2.93 (dt, J ¼ 10.2, 3.6 Hz, 1H), 2.54e2.43 (m, 1H),
2.43e2.32 (m, 1H), 2.19e2.00 (m, 2H), 1.91e1.80 (m, 1H), 1.79e1.68
(m, 1H), 1.64e1.40 (m, 4H), 1.28e1.19 (m, 1H). 13C NMR (75 MHz,
5.1.1.16. (6S,11aR,11bS)-3-((3-Chlorophenyl)(hydroxy)methyl)-
6,8,9,10,11,11a-hexahydro-2H-6,11b-methanofuro [2,3-c]pyrido [1,2-
a]azepin-2-one (3a-3). Yellow solid, yield 66%; 1H NMR
(300 MHz, CDCl3)
d 7.42 (s, 1H), 7.32e7.27 (m, 3H), 6.53e6.29 (m,
CDCl3)
d
173.18, 173.11, 164.21, 164.10, 148.49, 143.76, 143.71, 140.86,
2H), 5.63 (d, J ¼ 3.4 Hz, 1H), 3.82e3.73 (m, 1H), 2.93 (dt, J ¼ 10.5,
3.8 Hz, 1H), 2.54e2.43 (m, 1H), 2.44e2.33 (m, 1H), 2.15e2.00 (m,
1H), 1.93e1.80 (m, 1H), 1.79e1.68 (m, 1H), 1.64e1.49 (m, 4H),
132.59, 132.47, 129.76, 123.02, 121.16, 121.14, 120.65, 120.34, 117.52,
117.18, 88.88, 88.84, 68.36, 67.98, 63.07, 62.97, 58.84, 58.78, 48.73,
42.39, 42.26, 27.31, 27.20, 25.84, 25.73, 24.38, 24.26; ESI-LRMS m/z:
369.5 [MþH]þ; ESI-HRMS m/z: 369.1447 [MþH]þ, calcd for
1.28e1.20 (m, 1H). 13C NMR (75 MHz, CDCl3)
d
173.45, 173.34,
163.62, 163.49, 143.36, 140.25, 140.24,134.70, 130.10,128.36, 128.33,

158
W. Hou et al. / European Journal of Medicinal Chemistry 122 (2016) 149e163
126.52, 126.46, 124.63, 124.49, 120.88, 120.54, 88.79, 88.73, 68.83,
68.42, 63.13, 63.06, 58.84, 58.80, 48.78, 42.36, 42.31, 27.32, 27.26,
25.88, 25.81, 24.42, 24.36; ESI-LRMS m/z: 358.1 [MþH]þ; ESI-HRMS
m/z: 358.1203 [MþH]þ, calcd for C20H21ClNO3 358.1204.
a]azepin-2-one (4a-1-R). Yellow solid. m.p. 230.0e231.1 ꢁC; 1H
NMR (300 MHz, CDCl3)
d
8.25e8.16 (m, 2H), 7.62 (d, J ¼ 8.7 Hz, 2H),
6.46 (d, J ¼ 9.3 Hz, 1H), 6.37 (dd, J ¼ 9.3, 5.2 Hz, 1H), 5.78 (s, 1H, 16-
H), 3.80 (t, J ¼ 4.4 Hz, 1H), 23.0e2.83 (m, 1H), 2.48 (dd, J ¼ 9.2,
3.9 Hz, 1H), 2.44e2.32 (m, 1H), 2.06e1.96 (m, 1H), 1.89e1.79 (m,
5.1.1.17. (6S,11aR,11bS)-3-((3-Bromophenyl)(hydroxy)methyl)-
6,8,9,10,11,11a-hexahydro-2H-6,11b-methanofuro [2,3-c]pyrido [1,2-
a]azepin-2-one (3a-4). Yellow solid, yield 67%; 1H NMR
1H),1.75 (d, J ¼ 9.3 Hz,1H),1.63e1.44 (m, 4H),1.30e1.24 (m,1H). 13
C
NMR (75 MHz, CDCl3)
d 173.15, 164.15, 148.62, 147.69, 140.94,
127.09, 127.04, 124.05, 123.98, 120.51, 117.39, 88.89, 68.46, 63.09,
58.84, 48.78, 42.37, 27.32, 25.86, 24.40; ESI-LRMS m/z: 369.3
[MþH]þ; ESI-HRMS m/z: 369.1441 [MþH]þ, calcd for C20H21N2O5
369.1445.
(300 MHz, CDCl3)
d
7.59 (d, J ¼ 1.6 Hz, 1H), 7.47e7.41 (m, 1H), 7.35
(d, J ¼ 7.8 Hz, 1H), 7.22 (dd, J ¼ 7.8, 2.0 Hz, 1H), 6.56e6.31 (m, 2H),
5.64 (d, J ¼ 2.4 Hz, 1H), 3.81 (t, J ¼ 4.4 Hz, 1H), 3.05e2.84 (m, 1H),
2.58e2.41 (m, 2H), 2.27e2.13 (m, 1H), 1.86 (d, J ¼ 12.5 Hz, 1H),
1.82e1.73 (m, 1H), 1.67e1.54 (m, 4H), 1.30e1.21 (m, 1H). 13C NMR
5.1.1.22. (6S,11aR,11bS)-3-((4-Fluorophenyl)(hydroxy)methyl)-
6,8,9,10,11,11a-hexahydro-2H-6,11b-methanofuro [2,3-c]pyrido [1,2-
a]azepin-2-one (4a-2). Yellow solid, yield 80% (a:b ¼ 1:1); 1H
(75 MHz, CDCl3)
d 173.17, 173.15, 163.29, 163.16,143.53,143.51,
140.15, 140.02, 131.38, 131.15, 130.04, 129.47, 129.42, 125.13, 124.99,
120.66, 120.46, 118.33, 118.31, 88.74, 88.64, 68.86, 68.48, 63.07,
63.03, 59.06, 58.96, 48.72, 42.06,27.35, 27.30, 25.65, 24.56,
24.47.24.37; ESI-LRMS m/z: 404.3 [MþH]þ; ESI-HRMS m/z:
402.0710 [MþH]þ, calcd for C20H21BrNO3 402.0699.
NMR (300 MHz, CDCl3)
d
7.38 (dd, J ¼ 8.1, 5.7 Hz, 2H), 7.08e6.97 (m,
2H), 6.51e6.25 (m, 2H), 5.62 (d, J ¼ 5.4 Hz, 1H), 3.80e3.70 (m, 1H),
2.99e2.82 (m, 1H), 2.52e2.31 (m, 2H), 2.18e1.96 (m, 1H), 1.92e1.77
(m, 1H), 1.71 (dd, J ¼ 13.4, 9.3 Hz, 1H), 1.64e1.43 (m, 4H), 1.30e1.15
(m, 1H). 13C NMR (75 MHz, CDCl3)
d 173.49, 173.37, 164.13, 163.28,
5.1.1.18. 3-(Hydroxy((6S,11aR,11bS)-2-oxo-6,8,9,10,11,11a-hexahydro-
2H-6,11b-methanofuro [2,3-c]pyrido [1,2-a]azepin-3yl)methyl)ben-
zonitrile (3a-5). Yellow solid, yield 65%; 1H NMR (300 MHz, CDCl3)
163.21, 160.86, 139.87, 137.12, 128.26, 128.16, 128.13, 128.02, 121.03,
120.70, 118.45, 118.12, 115.78, 115.49, 88.66, 88.62, 68.92, 68.51,
63.08, 58.78, 58.75, 48.72, 42.20, 27.25, 27.17, 25.79, 25.72, 24.36,
24.27; ESI-LRMS m/z: 342.1 [MþH]þ; ESI-HRMS m/z: 342.1497
[MþH]þ, calcd for C20H21FNO3 342.1500.
d
7.77e7.64 (m, 2H), 7.59 (d, J ¼ 7.7 Hz, 1H), 7.47 (td, J ¼ 7.7, 3.9 Hz,
1H), 6.58e6.34 (m, 2H), 5.69 (s, 1H), 3.80 (dd, J ¼ 8.7, 4.2 Hz, 1H),
2.94 (dt, J ¼ 10.5, 3.6 Hz, 1H), 2.55e2.33 (m, 2H), 2.09 (dd, J ¼ 24.6,
10.8 Hz, 1H), 1.93e1.79 (m, 1H), 1.74 (dd, J ¼ 14.4, 9.3 Hz, 1H),
1.65e1.48 (m, 4H), 1.28e1.18 (m, 1H). 13C NMR (75 MHz, CDCl3)
5.1.1.23. (6S,11aR,11bS)-3-((4-Fluorophenyl)(hydroxy)methyl)-
6,8,9,10,11,11a-hexahydro-2H-6,11b-methanofuro [2,3-c]pyrido [1,2-
a]azepin-2-one (4a-2-a). Yellow solid. m.p. 170.1e171.2 ꢁC; 1H
d
173.20, 173.11, 164.02, 163.91, 143.12, 143.07, 140.72, 131.70, 130.91,
130.79, 129.82, 129.78, 129.57, 120.70, 120.36, 118.77, 117.71, 117.34,
112.72, 88.84, 88.80, 68.44, 68.01, 63.02, 62.95, 58.81, 58.76, 48.71,
42.38, 42.26, 27.30, 27.19, 25.83, 25.72, 24.36, 24.25; ESI-LRMS m/z:
349.2 [MþH]þ; ESI-HRMS m/z: 349.1550 [MþH]þ, calcd for
NMR (400 MHz, CDCl3)
d
7.40 (dd, J ¼ 8.6, 5.4 Hz, 2H), 7.09e7.03 (m,
2H), 6.39 (d, J ¼ 9.2 Hz, 1H), 6.32 (dd, J ¼ 9.3, 5.2 Hz, 1H), 5.65 (s, 1H,
16-H), 3.78 (t, J ¼ 4.6 Hz, 1H), 2.94 (dt, J ¼ 10.4, 3.8 Hz, 1H), 2.48 (dd,
J ¼ 9.3, 4.1 Hz, 1H), 2.46e2.38 (m, 1H), 2.14 (d, J ¼ 11.2 Hz, 1H),
1.91e1.84 (m, 1H), 1.73 (d, J ¼ 9.3 Hz, 1H), 1.69e1.48 (m, 4H), 1.26 (s,
1H).
C
21H21N2O3 349.1547.
5.1.1.19. (6S,11aR,11bS)-3-(Hydroxy(4-nitrophenyl)methyl)-
6,8,9,10,11,11a-hexahydro-2H-6,11b-methanofuro [2,3-c]pyrido [1,2-
a]azepin-2-one (4a-1). Yellow solid, yield 83% (R:S ¼ 1.81:1); 1H
5.1.1.24. (6S,11aR,11bS)-3-((4-Fluorophenyl)(hydroxy)methyl)-
6,8,9,10,11,11a-hexahydro-2H-6,11b-methanofuro [2,3-c]pyrido [1,2-
a]azepin-2-one (4a-2-b). Yellow solid. m.p. 189.0e189.2 ꢁC; 1H
NMR (300 MHz, CDCl3)
d 8.26e8.19 (m, 2H), 7.66e7.60 (m, 2H),
6.50e6.33 (m, 2H), 5.78 (s, 1H), 3.82 (t, J ¼ 4.4 Hz, 1H), 3.33e3.25
(m, 1H), 2.94 (dd, J ¼ 6.7, 4.0 Hz, 1H), 2.51 (dd, J ¼ 9.4, 4.2 Hz, 1H),
2.46e2.36 (m, 1H), 2.04 (d, J ¼ 4.2 Hz, 1H), 1.94e1.82 (m, 1H), 1.77
(d, J ¼ 9.3 Hz, 1H), 1.63e1.49 (m, 4H), 1.28e1.22 (m, 1H). 13C NMR
NMR (400 MHz, CDCl3)
d
7.40 (dd, J ¼ 8.6, 5.4 Hz, 2H), 7.12e7.03 (m,
2H), 6.33e6.28 (m, 2H), 5.67 (s, 1H, 16-H), 3.78 (t, J ¼ 4.0 Hz, 1H),
2.94 (dt, J ¼ 10.4, 3.7 Hz,1H), 2.49 (dd, J ¼ 9.2, 4.1 Hz,1H), 2.42e2.37
(m, 1H), 2.05 (dd, J ¼ 11.2, 2.6 Hz, 1H), 1.91e1.83 (m, 1H), 1.76 (d,
J ¼ 9.3 Hz, 1H), 1.64e1.50 (m, 4H), 1.26 (s, 1H).
(75 MHz, CDCl3)
d 173.19, 173.08, 164.12, 164.05, 148.70, 148.67,
147.58, 147.56, 140.83, 140.78, 127.04, 127.02, 123.97, 120.69, 120.35,
118.09, 118.08,117.66, 117.27, 68.35, 67.98, 62.96, 62.92, 58.87, 58.78,
48.69, 42.31, 42.13, 27.25, 27.06, 25.75, 25.55, 24.29, 24.06; ESI-
LRMS m/z: 369.3 [MþH]þ; ESI-HRMS m/z: 369.1441 [MþH]þ,
calcd for C20H21N2O5 369.1445.
5.1.1.25. (6S,11aR,11bS)-3-((4-Chlorophenyl)(hydroxy)methyl)-
6,8,9,10,11,11a-hexahydro-2H-6,11b-methanofuro [2,3-c]pyrido [1,2-
a]azepin-2-one (4a-3). Yellow solid, yield 78% (a:b ¼ 1.19:1); 1H
NMR (300 MHz, CDCl3) d 7.34e7.29 (m, 4H), 6.53e6.26 (m, 2H), 5.61
(d, J ¼ 4.4 Hz, 1H), 3.75 (dd, J ¼ 8.6, 4.1 Hz, 1H), 2.94e2.84 (m, 1H),
2.51e2.31 (m, 2H), 2.11e1.98 (m, 1H), 1.89e1.79 (m, 1H), 1.71 (dd,
J ¼ 14.3, 9.3 Hz, 1H), 1.62e1.47 (m, 4H), 1.28e1.22 (m, 1H). 13C NMR
5.1.1.20. (6S,11aR,11bS,16S)-3-(Hydroxy(4-nitrophenyl)methyl)-
6,8,9,10,11,11a-hexahydro-2H-6,11b-methanofuro [2,3-c]pyrido [1,2-
a]azepin-2-one (4a-1-S). Yellow solid. m.p. 216.2e217.3 ꢁC; 1H
(75 MHz, CDCl3)
d 173.26, 173.16, 163.29, 163.18, 139.90, 139.87,
NMR (300 MHz, CDCl3)
d
8.24e8.17 (m, 2H), 7.62 (d, J ¼ 8.6 Hz, 2H),
139.72, 139.69, 133.68, 133.64, 133.52, 128.73, 128.61, 127.62, 127.55,
127.52, 120.77, 120.48, 118.01, 117.72, 88.51, 88.49, 68.62, 68.22,
62.89, 62.87, 58.60, 58.57, 48.65, 48.56, 42.07, 42.05, 27.10, 27.02,
25.65, 25.59, 24.22, 24.14; ESI-LRMS m/z: 358.3 [MþH]þ; ESI-HRMS
m/z: 358.1213 [MþH]þ, calcd for C20H21ClNO3 358.1204.
6.53 (d, J ¼ 9.1 Hz, 1H), 6.37 (dd, J ¼ 9.3, 5.2 Hz, 1H), 5.77 (s, 1H, 16-
H), 3.79 (t, J ¼ 4.6 Hz, 1H), 2.91 (t, J ¼ 13.9 Hz, 1H), 2.52e2.37 (m,
2H), 2.11 (dd, J ¼ 10.9, 2.7 Hz, 1H), 1.94e1.80 (m, 1H), 1.70 (d,
J ¼ 9.3 Hz, 1H), 1.64e1.48 (m, 4H), 1.38e1.19 (m, 1H). 13C NMR
(75 MHz, CDCl3)
d 173.21, 164.03, 148.66, 147.65, 140.88, 127.04,
126.62, 124.01, 123.90, 120.30, 117.21, 88.89, 68.05, 62.98, 58.78,
48.74, 42.40, 27.35, 25.87, 24.42; ESI-LRMS m/z: 369.3 [MþH]þ; ESI-
HRMS m/z: 369.1441 [MþH]þ, calcd for C20H21N2O5 369.1445.
5.1.1.26. (6S,11aR,11bS)-3-((4-Chlorophenyl)(hydroxy)methyl)-
6,8,9,10,11,11a-hexahydro-2H-6,11b-methanofuro [2,3-c]pyrido [1,2-
a]azepin-2-one (4a-3-a). Yellow solid. m.p. 177.1e178.2 ꢁC; 1H
NMR (400 MHz, CDCl3)
2H), 6.42 (d, J ¼ 9.3 Hz, 1H), 6.32 (dd, J ¼ 9.3, 5.2 Hz, 1H), 5.64 (s, 1H,
d
7.37 (d, J ¼ 8.4 Hz, 2H), 7.33 (d, J ¼ 8.4 Hz,
5.1.1.21. (6S,11aR,11bS,16R)-3-(Hydroxy(4-nitrophenyl)methyl)-
6,8,9,10,11,11a-hexahydro-2H-6,11b-methanofuro [2,3-c]pyrido [1,2-
16-H), 3.77 (t, J ¼ 4.4 Hz, 1H), 2.93 (dt, J ¼ 10.4, 3.7 Hz, 1H), 2.47 (dd,

W. Hou et al. / European Journal of Medicinal Chemistry 122 (2016) 149e163
159
J ¼ 9.3, 4.0 Hz, 1H), 2.44e2.35 (m, 1H), 2.09 (dd, J ¼ 11.2, 2.4 Hz, 1H),
1.91e1.83 (m, 1H), 1.71 (d, J ¼ 9.3 Hz, 1H), 1.67e1.49 (m, 4H), 1.26 (s,
1H).
(400 MHz, CDCl3)
d
7.66 (dd, J ¼ 8.4, 3.1 Hz, 2H), 7.56 (dd, J ¼ 8.2,
3.7 Hz, 2H), 6.50 (d, J ¼ 9.2 Hz, 1H), 6.39 (d, J ¼ 4.2 Hz, 1H), 5.72 (s,
1H, 16-H), 3.82 (s, 1H), 2.95 (d, J ¼ 10.4 Hz, 1H), 2.50 (dd, J ¼ 9.3,
4.1 Hz, 1H), 2.20 (dd, J ¼ 16.3, 8.4 Hz, 1H), 2.01 (d, J ¼ 5.7 Hz, 1H),
1.87 (d, J ¼ 6.8 Hz,1H),1.74 (d, J ¼ 6.1 Hz,1H),1.64e1.54 (m, 4H),1.26
(s, 1H).
5.1.1.27. (6S,11aR,11bS)-3-((4-Chlorophenyl)(hydroxy)methyl)-
6,8,9,10,11,11a-hexahydro-2H-6,11b-methanofuro [2,3-c]pyrido [1,2-
a]azepin-2-one (4a-3-b). Yellow solid. m.p. 190.1e191.2 ꢁC; 1H
NMR (400 MHz, CDCl3)
d
7.37 (d, J ¼ 8.8 Hz, 2H), 7.34 (d, J ¼ 8.6 Hz,
5.1.1.33. 4-(Hydroxy((6S,11aR,11bS)-2-oxo-6,8,9,10,11,11a-hexahy-
dro-2H-6,11b-methanofuro [2,3-c]pyrido [1,2-a]azepin-3-yl)methyl)
benzonitrile (4a-5-b). Yellow solid. m.p. 192.9e193.8 ꢁC; 1H NMR
2H), 6.35e6.31 (m, 2H), 5.65 (s, 1H, 16-H), 3.83e3.76 (m, 1H), 2.94
(dt, J ¼ 10.3, 3.6 Hz, 1H), 2.49 (dd, J ¼ 9.2, 4.1 Hz, 1H), 2.43e2.34 (m,
1H), 2.04 (dd, J ¼ 11.2, 2.5 Hz, 1H), 1.90e1.81 (m, 1H), 1.75 (d,
J ¼ 9.3 Hz, 1H), 1.65e1.46 (m, 4H), 1.26 (s, 1H).
(400 MHz, CDCl3)
d
7.67 (dd, J ¼ 8.4, 2.0 Hz, 2H), 7.56 (dd, J ¼ 8.3,
4.0 Hz, 2H), 6.46 (d, J ¼ 9.3 Hz, 1H), 6.40e6.36 (m, 1H), 5.73 (s, 1H,
16-H), 3.80 (dd, J ¼ 9.8, 5.5 Hz, 1H), 2.95 (dt, J ¼ 10.3, 3.6 Hz, 1H),
2.48 (dd, J ¼ 8.9, 4.1 Hz,1H), 2.39 (dd, J ¼ 10.4, 7.3 Hz,1H), 2.14e2.07
(m, 1H), 1.92e1.83 (m, 1H), 1.73 (dd, J ¼ 16.0, 9.3 Hz, 1H), 1.69e1.48
(m, 4H), 1.26 (s, 1H).
5.1.1.28. (6S,11aR,11bS)-3-((4-Bromophenyl)(hydroxy)methyl)-
6,8,9,10,11,11a-hexahydro-2H-6,11b-methanofuro [2,3-c]pyrido [1,2-
a]azepin-2-one (4a-4). Yellow solid, yield 76% (a:b ¼ 1.36:1); 1H
NMR (300 MHz, CDCl3)
d 7.54e7.46 (m, 2H), 7.34e7.27 (m, 2H),
6.47e6.28 (m, 2H), 5.63 (d, J ¼ 4.1 Hz, 1H), 3.82e3.74 (m, 1H), 2.93
(dt, J ¼ 10.5, 3.7 Hz, 1H), 2.52e2.33 (m, 2H), 2.13e1.99 (m, 1H),
1.91e1.81 (m, 1H), 1.78e1.69 (m, 1H), 1.64e1.48 (m, 4H), 1.28e1.22
5.1.1.34. (6S,11aR,11bS)-3-(Hydroxy(4-(trifluoromethyl)phenyl)
methyl)-6,8,9,10,11,11a-hexahydro-2H-6,11b-methanofuro [2,3-c]pyr-
ido [1,2-a]azepin-2-one (4a-6). Yellow solid, yield 82% (a:b ¼ 1:2);
(m, J ¼ 6.0 Hz, 1H). 13C NMR (75 MHz, CDCl3)
d
173.45, 173.35,
1H NMR (300 MHz, CDCl3)
d
7.66e7.47 (m, 4H), 6.48 (dd, J ¼ 30.6,
163.39, 163.29, 147.06, 140.34, 140.15, 131.96, 131.95, 128.17, 128.06,
122.18, 122.13, 120.87, 120.57, 118.03, 117.77, 88.76, 88.71, 68.95,
68.58, 63.16, 63.11, 58.86, 58.82, 48.80, 42.35, 27.36, 27.29, 25.91,
25.85, 24.47, 24.41; ESI-LRMS m/z: 402.2 [MþH]þ; ESI-HRMS m/z:
402.0694 [MþH]þ, calcd for C20H21BrNO3 402.0699.
9.2 Hz,1H), 6.34 (dd, J ¼ 9.2, 5.3 Hz, 1H), 5.71 (d, J ¼ 2.4 Hz,1H), 3.77
(dd, J ¼ 9.0, 4.4 Hz, 1H), 2.98e2.84 (m, 1H), 2.53e2.32 (m, 2H),
2.16e1.99 (m, 1H), 1.85 (dd, J ¼ 16.9, 6.7 Hz, 1H), 1.72 (dd, J ¼ 14.4,
9.3 Hz, 1H), 1.65e1.43 (m, 4H), 1.28e1.18 (m, 1H). 13C NMR (75 MHz,
CDCl3)
d 173.38, 173.28, 163.83, 163.72, 145.44, 140.41, 130.46,
130.03, 126.63, 126.56, 125.80, 125.75, 125.70, 125.65, 120.80,
120.48, 117.97, 117.66, 88.82, 88.78, 68.77, 68.37, 63.08, 63.02, 58.85,
58.80, 48.84, 48.74, 27.32, 27.22, 25.86, 25.77, 24.40, 24.29; ESI-
LRMS m/z: 392.4 [MþH]þ; ESI-HRMS m/z: 392.1468 [MþH]þ,
calcd for C21H21F3NO3 392.1468.
5.1.1.29. (6S,11aR,11bS)-3-((4-Bromophenyl)(hydroxy)methyl)-
6,8,9,10,11,11a-hexahydro-2H-6,11b-methanofuro [2,3-c]pyrido [1,2-
a]azepin-2-one (4a-4-a). Yellow solid. m.p. 221.1e222.2 ꢁC; 1H
NMR (400 MHz, CDCl3)
d
7.51 (d, J ¼ 8.4 Hz, 2H), 7.33 (d, J ¼ 8.4 Hz,
2H), 6.45 (d, J ¼ 9.3 Hz, 1H), 6.35 (dd, J ¼ 9.3, 5.3 Hz, 1H), 5.65 (s, 1H,
16-H), 3.80 (t, J ¼ 4.8 Hz, 1H), 2.96 (dt, J ¼ 10.4, 3.7 Hz, 1H), 2.49 (dd,
J ¼ 9.3, 4.0 Hz, 1H), 2.43 (dd, J ¼ 9.3, 4.8 Hz, 1H), 2.12 (d, J ¼ 11.2 Hz,
1H), 1.89 (d, J ¼ 13.4 Hz, 1H), 1.74 (d, J ¼ 9.3 Hz, 1H), 1.68e1.50 (m,
4H),1.28 (s, 1H).
5.1.1.35. (6S,11aR,11bS)-3-(Hydroxy(4-(trifluoromethyl)phenyl)
methyl)-6,8,9,10,11,11a-hexahydro-2H-6,11b-methanofuro [2,3-c]pyr-
ido [1,2-a]azepin-2-one (4a-6-a). Yellow solid. m.p. 160.7e161.9 ꢁC;
1H NMR (300 MHz, CDCl3)
d
7.59 (dd, J ¼ 20.1, 8.4 Hz, 4H), 6.47 (d,
J ¼ 8.7 Hz, 1H), 6.35 (dd, J ¼ 9.3, 5.2 Hz, 1H), 5.73 (s, 1H, 16-H), 3.78
(t, J ¼ 4.6 Hz, 1H), 2.94 (dt, J ¼ 10.5, 3.7 Hz, 1H), 2.55e2.32 (m, 1H),
2.11 (dd, J ¼ 11.1, 2.7 Hz, 1H), 1.95e1.80 (m, 1H), 1.72 (d, J ¼ 9.3 Hz,
1H), 1.67e1.44 (m, 1H), 1.34e1.13 (m, 1H). 13C NMR (75 MHz, CDCl3)
5.1.1.30. (6S,11aR,11bS)-3-((4-Bromophenyl)(hydroxy)methyl)-
6,8,9,10,11,11a-hexahydro-2H-6,11b-methanofuro [2,3-c]pyrido [1,2-
a]azepin-2-one (4a-4-b). Yellow solid. m.p. 196.0e197.0 ꢁC; 1H
NMR (400 MHz, CDCl3)
d
7.49 (d, J ¼ 8.4 Hz, 2H), 7.30 (d, J ¼ 8.4 Hz,
d 173.15, 164.15, 148.62, 147.69, 140.94, 127.09, 127.04, 124.05,
2H), 6.36 (d, J ¼ 9.3 Hz, 1H), 6.31 (dd, J ¼ 9.3, 5.3 Hz, 1H), 5.63 (s, 1H,
16-H), 3.78 (t, J ¼ 4.4 Hz, 1H), 2.93 (dt, J ¼ 10.4, 3.7 Hz, 1H), 2.48 (dd,
J ¼ 9.3, 4.0 Hz, 1H), 2.43e2.33 (dd, J ¼ 9.3, 4.8 Hz, 1H), 2.03 (dd,
J ¼ 11.2, 2.6 Hz, 1H), 1.85 (d, J ¼ 13.4 Hz, 1H), 1.74 (d, J ¼ 9.3 Hz, 1H),
1.64e1.44 (m, 4H), 1.25 (s, 1H).
123.98, 120.51, 117.39, 88.89, 68.46, 63.09, 58.84, 48.78, 42.37, 27.32,
25.86, 24.40; ESI-LRMS m/z: 392.4 [MþH]þ; ESI-HRMS m/z:
392.1468 [MþH]þ, calcd for C21H21F3NO3 392.1468.
5.1.1.36. (6S,11aR,11bS)-3-(Hydroxy(4-(trifluoromethyl)phenyl)
methyl)-6,8,9,10,11,11a-hexahydro-2H-6,11b-methanofuro [2,3-c]pyr-
ido [1,2-a]azepin-2-one (4a-6-b). Yellow solid. m.p. 186.0e187.2 ꢁC;
5.1.1.31. 4-(Hydroxy((6S,11aR,11bS)-2-oxo-6,8,9,10,11,11a-hexahydro-
2H-6,11b-methanofuro [2,3-c]pyrido [1,2-a]azepin-3-yl)methyl)ben-
zonitrile (4a-5). Yellow solid, yield 75% (a:b ¼ 1:1.25); 1H NMR
1H NMR (300 MHz, CDCl3)
d
7.59 (dd, J ¼ 23.8, 8.2 Hz, 4H),
6.41e6.30 (m, 2H), 5.74 (s, 1H,16-H), 3.80 (t, J ¼ 3.3 Hz,1H), 2.94 (dt,
J ¼ 10.5, 3.7 Hz,1H), 2.49 (dt, J ¼ 10.1, 5.1 Hz,1H), 2.45e2.32 (m, 1H),
2.11e2.00 (m, 1H), 1.93e1.80 (m, 1H), 1.76 (d, J ¼ 9.4 Hz, 1H),
1.64e1.49 (m, 4H), 1.30e1.18 (m, 1H). 13C NMR (75 MHz, CDCl3)
(300 MHz, CDCl3)
d 7.65e7.58 (m, 2H), 7.58e7.49 (m, 2H), 6.50 (dd,
J ¼ 33.3, 9.3 Hz, 1H), 6.35 (dd, J ¼ 9.3, 5.2 Hz, 1H), 5.70 (d, J ¼ 2.5 Hz,
1H), 3.83e3.73 (m, 1H), 2.98e2.86 (m, 1H), 2.51e2.32 (m, 2H),
2.16e1.96 (m, 1H), 1.91e1.77 (m, 1H), 1.70 (dd, J ¼ 18.4, 9.3 Hz, 1H),
1.61e1.47 (m, 4H), 1.31e1.25 (m, J ¼ 3.6 Hz, 1H). 13C NMR (75 MHz,
d
173.21, 164.03, 148.66, 147.65, 140.88, 127.04, 126.62, 124.01,
123.90, 120.30, 117.21, 88.89, 68.05, 62.98, 58.78, 48.74, 42.40, 27.35,
25.87, 24.42; ESI-LRMS m/z: 392.4 [MþH]þ; ESI-HRMS m/z:
392.1468 [MþH]þ, calcd for C21H21F3NO3 392.1468.
CDCl3) d 172.96,172.81,171.50,171.37,163.78,163.64,146.85,146.82,
140.36, 140.14, 132.44, 126.86, 121.10, 120.65, 118.67, 117.69, 111.46,
88.64, 88.55, 77.16, 68.40, 67.97, 62.69, 62.60, 58.94, 58.77, 48.44,
48.32, 41.88, 41.54, 26.80, 26.42, 25.23, 24.79, 23.72, 23.20; ESI-
LRMS m/z: 349.2 [MþH]þ; ESI-HRMS m/z: 349.1555 [MþH]þ,
calcd for C21H21N2O3 349.1547.
5.1.1.37. (6S,11aR,11bS)-3-(Hydroxy(4-methoxyphenyl)methyl)-
6,8,9,10,11,11a-hexahydro-2H-6,11b-methanofuro [2,3-c]pyrido [1,2-
a]azepin-2-one (4b-2). Yellow sticky solid, yield 69%; 1H NMR
(300 MHz, CDCl3)
d 7.36e7.27 (m, 3H), 6.91e6.82 (m, 3H),
5.1.1.32. 4-(Hydroxy((6S,11aR,11bS)-2-oxo-6,8,9,10,11,11a-hexahy-
dro-2H-6,11b-methanofuro [2,3-c]pyrido [1,2-a]azepin-3-yl)methyl)
benzonitrile (4a-5-a). Yellow solid. m.p. 179.1e180.2 ꢁC; 1H NMR
6.49e6.32 (m, 1H), 6.31e6.23 (m, 1H), 5.59 (d, J ¼ 6.7 Hz, 1H),
3.80e3.76 (m, 4H), 2.89 (d, J ¼ 3.2 Hz, 1H), 2.48e2.41 (m, 1H),
1.89e1.81 (m, 1H), 1.77e1.67 (m, 1H), 1.62e1.49 (m, 4H), 1.29e1.23

160
W. Hou et al. / European Journal of Medicinal Chemistry 122 (2016) 149e163
(m, 1H). 13C NMR (75 MHz, CDCl3)
d
173.52, 173.40, 167.84, 162.93,
5.1.1.42. (6S,11aR,11bS,16R)-3-((2,4-Dinitrophenyl)(hydroxy)methyl)-
162.84, 159.47, 159.43, 139.34, 133.43, 132.46, 130.96, 128.85, 127.84,
127.68, 121.32, 121.01, 118.84, 118.55, 114.11, 88.51, 88.46, 69.32,
63.12, 58.76, 55.33, 48.73, 42.20, 27.20, 25.75, 24.33; ESI-LRMS m/z:
340.3 [MþH]þ; ESI-HRMS m/z: 340.1542 [MþH]þ, calcd for
6,8,9,10,11,11a-hexahydro-2H-6,11b-methanofuro [2,3-c]pyrido [1,2-
a]azepin-2-one (5a-1-R). Brown solid. m.p. 250.7e251.8 ꢁC; 1H
NMR (300 MHz, CDCl3)
d
8.77 (d, J ¼ 2.3 Hz, 1H), 8.53 (dd, J ¼ 8.7,
2.3 Hz, 1H), 8.28 (d, J ¼ 8.7 Hz, 1H), 6.41 (d, J ¼ 3.0 Hz, 2H), 6.33 (s,
1H, 16-H), 3.84 (dd, J ¼ 6.6, 3.4 Hz, 1H), 2.95 (dt, J ¼ 10.7, 3.6 Hz, 1H),
2.51 (dd, J ¼ 9.3, 4.1 Hz, 1H), 2.47e2.34 (m, 1H), 2.03 (dd, J ¼ 10.7,
2.8 Hz, 1H), 1.91e1.81 (m, 1H), 1.78 (d, J ¼ 9.3 Hz, 1H), 1.60e1.50 (m,
4H), 1.25 (s, 1H).
C
21H24NO4 340.1543.
5.1.1.38. (6S,11aR,11bS)-3-(Hydroxy(4-(methylthio)phenyl)methyl)-
6,8,9,10,11,11a-hexahydro-2H-6,11b-methanofuro [2,3-c]pyrido [1,2-
a]azepin-2-one (4b-3). Yellow sticky solid, yield 68%; 1H NMR
5.1.1.43. (6S,11aR,11bS,16S)-3-((2,4-Dinitrophenyl)(hydroxy)methyl)-
6,8,9,10,11,11a-hexahydro-2H-6,11b-methanofuro [2,3-c]pyrido [1,2-
a]azepin-2-one (5a-1-S). Brown solid. m.p. 277.1e278.2 ꢁC; 1H
(300 MHz, CDCl3)
d
7.33 (d, J ¼ 8.4 Hz, 2H), 7.25e7.19 (m, 2H),
6.50e6.26 (m, 2H), 5.61 (d, J ¼ 5.7 Hz, 1H), 3.75 (d, J ¼ 4.9 Hz, 1H),
2.95e2.90 (m, 1H), 2.50e2.43 (m, 4H), 2.14e2.01 (m, 1H), 2.15e2.01
(m, 1H), 1.90e1.80 (m, 1H), 1.78e1.68 (m, 1H), 1.62e1.47 (m, 4H),
NMR (300 MHz, CDCl3)
d
8.77 (d, J ¼ 2.3 Hz, 1H), 8.53 (dd, J ¼ 8.7,
1.27e1.21 (m,1H). 13C NMR (75 MHz, CDCl3)
d 173.55,173.45,163.19,
2.3 Hz,1H), 8.28 (d, J ¼ 8.7 Hz,1H), 6.42 (dd, J ¼ 9.2, 5.2 Hz,1H), 6.35
(s, 1H), 6.31 (s, 1H, 16-H) 3.84 (t, J ¼ 4.4 Hz, 1H), 2.96 (dt, J ¼ 10.5,
3.6 Hz, 1H), 2.48 (dd, J ¼ 9.5, 4.2 Hz, 1H), 2.43 (s, 1H), 2.10 (d,
J ¼ 9.2 Hz, 1H), 1.89 (d, J ¼ 14.1 Hz, 1H), 1.74 (d, J ¼ 9.4 Hz, 1H),
1.65e1.54 (m, 4H), 1.25 (s, 1H).
163.09, 139.78, 139.76, 138.59, 138.50, 138.02, 127.01, 126.87, 126.71,
126.69, 121.11, 121.04, 121.00, 120.84, 120.80, 118.36, 118.09, 88.66,
88.62, 69.24, 68.83, 63.14, 58.79, 48.77, 42.27, 27.30, 27.26, 25.85,
25.82, 24.42, 24.39, 15.79; ESI-LRMS m/z: 370.4 [MþH]þ; ESI-HRMS
m/z: 370.1479 [MþH]þ, calcd for C21H24NO3S 370.1471.
5.1.1.44. (6S,11aR,11bS)-3-((2,4-Difluorophenyl)(hydroxy)methyl)-
6,8,9,10,11,11a-hexahydro-2H-6,11b-methanofuro [2,3-c]pyrido [1,2-
a]azepin-2-one (5a-2). Yellow solid, yield 85% (a:b ¼ 1:1.24); 1H
5.1.1.39. (6S,11aR,11bS)-3-(Hydroxy(p-tolyl)methyl)-6,8,9,10,11,11a-
hexahydro-2H-6,11b-methanofuro [2,3-c]pyrido [1,2-a]azepin-2-one
(4b-4). Yellow solid, yield 65%; 1H NMR (300 MHz, CDCl3)
NMR (300 MHz, CDCl3)
d
7.67e7.48 (m, 1H), 6.91 (t, J ¼ 8.4 Hz, 1H),
6.78 (t, J ¼ 9.6 Hz, 1H), 6.46e6.27 (m, 2H), 5.87 (d, J ¼ 4.9 Hz, 1H),
3.78 (dd, J ¼ 7.8, 3.8 Hz, 1H), 3.31 (s, 1H), 2.93 (d, J ¼ 10.5 Hz, 1H),
2.45 (dt, J ¼ 37.5, 14.2 Hz, 2H), 2.16e2.00 (m, 1H), 1.93e1.68 (m, 2H),
1.68e1.45 (m, 4H), 1.33e1.22 (m, 1H). 13C NMR (75 MHz, CDCl3)
d
7.33e7.24 (m, 2H), 7.22e7.08 (m, 2H), 6.43 (dd, J ¼ 30.4, 9.3 Hz,
1H), 6.33e6.20 (m, 1H), 5.64e5.50 (m, 1H), 3.74 (s, 1H), 3.38e3.22
(m, 1H), 2.98e2.78 (m, 1H), 2.49e2.38 (m, 1H), 2.33 (s, 3H),
2.15e1.97 (m, 1H), 1.95e1.67 (m, 2H), 1.64e1.36 (m, 4H), 1.30e1.17
d
173.44, 173.35, 163.54, 163.39, 140.17, 140.04, 129.25, 129.18,
(m, 1H). 13C NMR (75 MHz, CDCl3)
d 173.36, 173.25, 162.89, 162.77,
129.12, 129.05, 124.39, 124.26, 120.55, 120.40, 116.71, 116.59, 111.86,
111.58, 104.36, 104.34, 104.02, 104.00, 103.69, 103.67, 88.77, 88.76,
63.10, 62.91, 58.83, 58.81, 48.76, 48.72, 42.36, 42.14, 27.26, 27.18,
25.77, 25.73, 24.36, 24.27; ESI-LRMS m/z: 360.4 [MþH]þ; ESI-HRMS
m/z: 360.1407 [MþH]þ, calcd for C20H20F2NO3 360.1406.
139.25, 138.45, 138.13, 137.73, 137.65, 137.48, 129.25, 129.16, 126.25,
126.09, 121.10, 120.79, 118.52, 118.27, 88.36, 88.31, 68.85, 68.56,
62.94, 59.67, 58.57, 48.62, 48.57, 42.03, 42.00, 27.07, 27.05, 25.73,
25.62, 24.20, 21.08, 21.03; ESI-LRMS m/z: 338.1 [MþH]þ; ESI-HRMS
m/z: 338.1744 [MþH]þ, calcd for C21H24NO3 338.1751.
5.1.1.45. (6S,11aR,11bS)-3-((2,4-Difluorophenyl)(hydroxy)methyl)-
6,8,9,10,11,11a-hexahydro-2H-6,11b-methanofuro [2,3-c]pyrido [1,2-
a]azepin-2-one (5a-2-a). Yellow solid. m.p. 158.1e159.1 ꢁC; 1H
5.1.1.40. (6S,11aR,11bS)-3-(Hydroxy(phenyl)methyl)-6,8,9,10,11,11a-
hexahydro-2H-6,11b-methanofuro [2,3-c]pyrido [1,2-a]azepin-2-one
(4b-5). Yellow solid, yield 67%; 1H NMR (300 MHz, CDCl3)
NMR (300 MHz, CDCl3)
d
7.56 (td, J ¼ 8.5, 6.9 Hz, 1H), 6.92 (td,
d
7.43e7.29 (m, 5H), 6.46e6.25 (m, 2H), 5.66 (d, J ¼ 5.6 Hz, 1H), 3.75
J ¼ 8.2, 1.6 Hz,1H), 6.79 (ddd, J ¼ 11.0, 8.8, 2.4 Hz,1H), 6.50e6.31 (m,
2H), 5.88 (s, 1H, 16-H), 3.81 (d, J ¼ 3.2 Hz, 1H), 2.93 (d, J ¼ 10.2 Hz,
1H), 2.50 (dd, J ¼ 9.0, 4.0 Hz, 2H), 2.10 (d, J ¼ 12.4 Hz, 1H), 1.91e1.73
(m, 2H), 1.68e1.45 (m, 4H), 1.24 (s, 1H).
(t, J ¼ 4.1 Hz, 1H), 2.92 (dt, J ¼ 10.4, 3.8 Hz, 1H), 2.49e2.32 (m, 2H),
2.10 (ddd, J ¼ 20.3, 11.0, 2.3 Hz, 1H), 1.88e1.80 (m, 1H), 1.74 (dd,
J ¼ 11.1, 9.6 Hz, 1H), 1.64e1.48 (m, 4H), 1.31e1.21 (m, 1H). 13C NMR
(75 MHz, CDCl3)
d 173.41, 173.29, 163.02, 162.92, 141.04, 139.43,
128.65, 128.63, 128.09, 128.03, 126.33, 126.19, 126.16, 121.02, 120.67,
118.45, 118.15, 88.47, 88.42, 69.40, 68.97, 62.96, 62.95, 58.67, 58.65,
48.59, 42.05, 27.09, 27.03, 25.62, 25.57, 24.18, 24.12; ESI-LRMS m/z:
324.4 [MþH]þ; ESI-HRMS m/z: 324.1592 [MþH]þ, calcd for
5.1.1.46. (6S,11aR,11bS)-3-((2,4-Difluorophenyl)(hydroxy)methyl)-
6,8,9,10,11,11a-hexahydro-2H-6,11b-methanofuro [2,3-c]pyrido [1,2-
a]azepin-2-one (5a-2-b). Yellow solid. m.p. 149.2e150.3 ꢁC; 1H
NMR (300 MHz, CDCl3)
d
7.59 (dt, J ¼ 8.5, 4.3 Hz, 1H), 6.98e6.88 (m,
C
20H21NO3 324.1594.
1H), 6.78 (ddd, J ¼ 11.0, 8.8, 2.5 Hz, 1H), 6.39e6.28 (m, 2H), 5.89 (s,
1H, 16-H), 3.77 (t, J ¼ 4.0 Hz, 1H), 2.93 (dt, J ¼ 10.5, 3.7 Hz, 1H), 2.45
(dd, J ¼ 9.3, 4.1 Hz, 1H), 2.41e2.32 (m, 1H), 2.07 (dd, J ¼ 11.1, 2.6 Hz,
1H), 1.91e1.82 (m, 1H), 1.72 (d, J ¼ 9.2 Hz, 1H), 1.66e1.44 (m, 4H),
1.23 (s, 1H).
5.1.1.41. (6S,11aR,11bS)-3-((2,4-Dinitrophenyl)(hydroxy)methyl)-
6,8,9,10,11,11a-hexahydro-2H-6,11b-methanofuro [2,3-c]pyrido [1,2-
a]azepin-2-one (5a-1). Brown solid, yield 85% (R:S ¼ 1.11:1); 1H
NMR (300 MHz, CDCl3)
d
8.75 (d, J ¼ 2.3 Hz, 1H), 8.52 (dd, J ¼ 8.7,
2.3 Hz, 1H), 8.29 (d, J ¼ 8.7 Hz, 1H), 6.45e6.23 (m, 3H), 3.82 (t,
J ¼ 4.6 Hz, 1H), 3.47 (s, 1H), 2.94 (dt, J ¼ 10.5, 3.9 Hz, 1H), 2.94 (dt,
J ¼ 10.5, 3.9 Hz, 1H), 2.52e2.32 (m, 2H), 2.04 (d, J ¼ 10.6 Hz, 1H),
1.93e1.82 (m, 1H), 1.73 (d, J ¼ 9.3 Hz, 1H), 1.65e1.48 (m, 4H),
5.1.2. General procedure of the preparation of 4b, 4b-1
p-Hydroxybenzaldehyde (2.45 mmol, 299 mg) dissolved in dry
DCM (5 mL) and acetyl chloride (2.95 mmol, 232 mg) was added in
a dry anaerobic double-neck flask at 0 ꢁC. After stirring for 1 h, we
obtained 4-(acetyloxy)benzaldehyde (395 mg). Securinine
(0.69 mmol, 150 mg) and 4-(acetyloxy)benzaldehyde (1.04 mmol,
170 mg) were added into a dry anaerobic double-neck flask con-
taining 5 mL dry DCM at 2 ꢁC. TiCl4 (1.248 mmol) was added after
the reactants dissolved. The reaction was monitored by TLC on an
hourly basis and terminated after 12 h. Then saturated aqueous
sodium bicarbonate solution was added until the pH reached 9.0.
1.27e1.22 (m, 1H). 13C NMR (75 MHz, CDCl3)
d 172.88, 165.43,
165.21, 147.59, 147.57, 147.35, 143.09, 142.93, 141.59, 141.56, 131.05,
130.90, 127.46, 127.35, 120.16, 120.13, 119.91, 115.68, 115.33, 88.91,
88.87, 63.67, 63.56, 63.02, 62.77, 58.88, 58.80, 48.71, 42.55, 42.10,
27.19, 27.09, 25.77, 25.65, 24.32, 24.21; ESI-LRMS m/z: 414.4
[MþH]þ; ESI-HRMS m/z: 414.1295 [MþH]þ, calcd for C20H20N3O7
414.1296.

W. Hou et al. / European Journal of Medicinal Chemistry 122 (2016) 149e163
161
The mixture was filtered, and extracted with DCM. The DCM layer
was dried over anhydrous Na2SO4, filtered and concentrated to
obtain a mixture residue, which was purified on a silica gel column
(DCM:MeOH ¼ 200:1) to give 4b (80 mg). Then 4b (30 mg) was
dissolved in MeOH (2 mL) and sodium bicarbonate (1 mmol, 84 mg)
was added at 25 ꢁC. After 1 h, the mixture were filtered and the
filter liquor was evaporated to give a residue which was subjected
to silica gel chromatography (DCM:MeOH ¼ 200:1) to give 4b-1
(27 mg).
of 2a-1-R, 2a-1-S, 3a-1-R, 3a-1-S, 4a-1-R, 4a-1-S, 5a-1-R and 5a-1-
S were confirmed. Diastereomers showing a positive cotton effect
tendency from 250 to 300 nm were assigned as S configuration at
the C-16. On the contrary, it was inferred as R configuration. The
absolute structures of diastereomers 3a-2-a, 3a-2-b, 4a-2-a, 4a-2-
b, 4a-4-a, 4a-4-b, 4a-6-a, 4a-6-b, 5a-2-a and 5a-2-b, which do not
show obvious different cotton effect tendency around 250 and
300 nm, thus cannot be identified this time.
5.2. Bioassays
5.1.2.1. 4-(Hydroxy((6S,11aR,11bS)-2-oxo-6,8,9,10,11,11a-hexahydro-
2H-6,11b-methanofuro
phenyl acetate (4b). Yellow solid, yield 78%; 1H NMR (300 MHz,
CDCl3)
[2,3-c]pyrido
[1,2-a]azepin-3-yl)methyl)
5.2.1. Topo I inhibitory activity
Relaxed pBR322 DNA derived from supercoiled pBR322 DNA
(TaKaRa, Kyoto, Japan) was measured to reflect inhibition of the
Topo I (TaKaRa, Kyoto, Japan) catalytic ability by test compounds.
Camptothecin was used as a positive control. The reaction solution
was prepared according to the supplier’s instructions, and incu-
bated at 37 ꢁC for 30 min. A dye solution containing 0.25% bro-
mophenol blue, 0.25% xylene cyanol ff and 40% glycerol was added
to end the reaction. The mixtures were added to 1% agarose gel and
subjected to electrophoresis for 40 min in 1 ꢂ TAE buffer (40 mM
Tris-acetate, 2 mM EDTA, 19.9 mM HOAc). Then the gels were
d
7.43 (d, J ¼ 8.4 Hz, 2H), 7.07 (dt, J ¼ 4.9, 2.8 Hz, 2H),
6.53e6.26 (m, 2H), 5.63 (d, J ¼ 5.9 Hz, 1H), 3.80e3.70 (m, 1H), 2.92
(dt, J ¼ 10.4, 3.7 Hz, 1H), 2.53e2.32 (m, 2H), 2.28 (d, J ¼ 0.9 Hz, 3H),
2.15e2.00 (m, 1H), 1.92e1.80 (m, 1H), 1.79e1.69 (m, 1H), 1.67e1.47
(m, 4H), 1.29e1.17 (m, 1H). 13C NMR (75 MHz, CDCl3)
d 173.46,
173.38,169.59,163.35,163.24,150.42,150.40,139.90,139.85,138.92,
138.90, 127.59, 127.48, 121.90, 121.04, 120.77, 118.23, 117.99, 88.67,
88.63, 69.07, 68.70, 63.13, 63.08, 58.78, 58.76, 48.78, 48.77, 42.31,
27.32, 27.30, 25.89, 24.44, 21.24; ESI-LRMS m/z: 382.3 [MþH]þ; ESI-
HRMS m/z: 382.1652 [MþH]þ, calcd for C22H24NO5 382.1649.
stained for 30 min in 200 mL 1 X TAE buffer containing 5 mg/ml EB,
and washed for 20 min in 200 mL 1 ꢂ TAE buffer. DNA bands were
visualized by transillumination with UV light and photographed
with an Alpha Innotech digital imaging system.
5.1.2.2. (6S,11aR,11bS)-3-(Hydroxy(4-hydroxyphenyl)methyl)-
6,8,9,10,11,11a-hexahydro-2H-6,11b-methanofuro [2,3-c]pyrido [1,2-
a]azepin-2-one (4b-1). Yellow solid, yield 90%; 1H NMR
(300 MHz, DMSO-d6)
d
7.16 (d, J ¼ 8.5 Hz,1H), 7.11 (d, J ¼ 8.5 Hz,1H),
5.2.2. Cell growth inhibition assay
6.89 (dd, J ¼ 25.8, 9.1 Hz,1H), 6.79e6.66 (m, 2H), 6.50e6.34 (m,1H),
5.69 (dd, J ¼ 7.7, 4.0 Hz, 1H), 5.37 (dd, J ¼ 11.7, 3.9 Hz, 1H), 3.88e3.78
(m, 1H), 3.17 (d, J ¼ 5.1 Hz, 1H), 3.06e2.84 (m, 1H), 2.58e2.43 (m,
1H), 2.38e2.24 (m, 2H), 1.86e1.76 (m, 1H), 1.64 (dd, J ¼ 21.2, 9.0 Hz,
1H), 1.56e1.29 (m, 4H), 1.27e1.11 (m, 1H). 13C NMR (75 MHz, DMSO-
The growth inhibitory effect of securinine derivatives toward
four different human cancer cell lines, A549 (adenocarcinomic
human alveolar basal epithelial cell line), HeLa (human cervix tu-
mor cell line), HepG2 (human liver tumor cell), SH-SY5Y (human
neuroblastoma cell line) and L02 cell line (a healthy mammalian
cell line) were assessed with a classical MTT assay [22]. The cells
were plated in a density of 5 ꢂ 103 per well in 96-well microplates,
d6)
d 172.10, 171.96, 162.68, 162.47, 156.76, 156.74, 139.05, 138.85,
133.14, 133.08, 127.79, 127.64, 121.69, 121.61, 119.72, 119.56, 115.07,
115.04, 87.28, 68.19, 67.62, 62.99, 57.93, 57.89, 48.71, 48.31, 48.18,
42.11, 41.93, 27.24, 25.71, 25.65, 24.47; ESI-LRMS m/z: 340.3
[MþH]þ; ESI-HRMS m/z: 340.1542 [MþH]þ, calcd for C20H22NO4
340.1543.
and incubated overnight in 100 ml of a medium containing 10% Fetal
Bovine Serum (Hyclone, USA). On the following day, the culture
medium was replaced by fresh medium containing securinine de-
rivatives with increasing concentrations (1, 12.5, 25, 50, 75, 100 mM)
for a cultivation lasting 72 h. Then 20 mL of 5 mg/mL MTT solution
were added into each well and after an incubation of 4 h at 37 ꢁC,
the culture medium was removed, and 120 mL DMSO was added.
For a better dissolution of the formazan dye, the 96 well micro-
plates were shaken for 10 min, and the absorption at 570 nm was
detected using a microplate-reader (Bio-Tek). Parallel triplicate
repetition for each dose was made. The IC50 values were elicited
from the mean light absorption value from the triplicate test versus
compounds concentration curves.
5.1.3. X-ray crystallographic and circular dichroism spectra analysis
Upon crystallization from MeOH using the vapor diffusion
method, crystal of 2a-2-S were obtained. Data were collected using
a Sapphire CCD with a graphite monochromated Cu K
a radiation
(
l
¼
1.54184 Å) at 150.00(2) K. Crystal data: C20H20FNO3,
M ¼ 341.37, monoclinic, space group P21; unit cell dimensions were
determined to be a ¼ 8.0898(1) Å, b ¼ 12.3867(1) Å, c ¼ 8.5243(1) Å,
b
¼ 104.627(1)ꢁ, V ¼ 826.501(16) Å3, Z ¼ 2, Dx ¼ 1.372 g/cm3, F
(000) ¼ 360,
m
(Cu Ka) ¼ 0.821 mm-1. 11365 reflections were
collected until qmax ¼ 66.89ꢁ, in which independent unique 2840
5.2.3. DNA-insertion assay
This assays was similar to the Topo I inhibitory assay, with a
minor variation. Calf thymus Topo I (20 units) (TaKaRa, Kyoto,
reflections were observed [F2 > 4
s
(F2)]. The structure was solved
and refined using the OLEX2 program. In the structure refinements,
nonhydrogen atoms were placed on the geometrically ideal posi-
tions by the “ride on” method. Hydrogen atoms bonded to oxygen
were located by the structure factors with isotropic temperature
factors. The final refinement gave R ¼ 0.0288, RW ¼ 0.0297, and
S ¼ 1.088. CCDC 1480589.
Japan), 0.1
Japan), and 100
bromide) were added in a total of 20
m
g negatively supercoiled pBR322DNA (TaKaRa, Kyoto,
M securinine derivatives (or 25 M ethidium
L of Topo I buffer (50 mM
m
m
m
Tris-HCl, pH 7.5, 10 mM MgCl2, 50 mM KCl, 0.1 mM EDTA, 0.5 mM
DTT, and 30 mg/mL bovine serum albumin). After incubation for
For CD spectrum analysis, pure diastereomers samples were
dissolved in methyl alcohol at the concentration of 0.5 mg/ml.
Spectra were recorded on a Chirascan spectrometer (Applied Pho-
tophysics) with a 0.1-cm cell at a scan of 50 nm/min with a response
time of 1 s.
30 min at 37 ꢁC, the samples were mixed with 4
mL of 6 x loading
buffer (0.25% bromophenol blue, xylene cyanol ff 0.25% and 40%
glycerol), and subjected to electrophoresis in a 1% agarose gel in 1 ꢂ
TAE buffer (40 mM Tris-acetate, 2 mM EDTA, 19.9 mM HOAc) for
40 min. Then the gel was stained in 200 mL of 1 ꢂ TAE buffer
Based on the absolute structure of 2a-2-S and CD spectra (in
supplementary material) of 2a-1-R, 2a-1-S, 2a-2-R, 2a-2-S, 3a-1-R,
3a-1-S, 4a-1-R, 4a-1-S, 5a-1-R and 5a-1-S, the C-16 configurations
comprising 0.5 mg/mL ethidium bromide for 30 min and embathed
in 200 mL of 1 ꢂ TAE buffer for another 20 min. DNA bands were
visualized by transillumination with UV light, photographed by

162
W. Hou et al. / European Journal of Medicinal Chemistry 122 (2016) 149e163
Alpha Innotech digital imaging system.
Acknowledges
We sincerely thank the Natural Science Foundation of China
(81273362) for financial support of this study.
5.2.4. Topo I-mediated DNA cleavage assay
Calf thymus Topo I (30 units), 0.5 g negatively supercoiled
pBR322DNA, and 25e200 M securinine derivatives (or 25e50
camptothecin) were added to 40 L of Topo I buffer (50 mM Tris-
m
m
mM
Appendix A. Supplementary data
m
HCl, pH 7.5, 50 mM KCl, 10 mM MgCl2, 0.5 mM DTT, 0.1 mM
EDTA, and 30 mg/mL bovine serum albumin). After incubating for
30 min at 37 ꢁC, cleavage intermediates were trapped by adding 1%
SDS to terminate the reaction, followed by 0.8 mg/mL Proteinase K
(pH 8.0), and reactions were incubated for 10 min at 50 ꢁC to digest
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.06.021.
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